CN117871216B - Three-dimensional visualization method for glomerular fissure membrane in paraffin specimen - Google Patents
Three-dimensional visualization method for glomerular fissure membrane in paraffin specimen Download PDFInfo
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Abstract
The invention relates to the technical field of tissue fluorescent staining, in particular to a three-dimensional visualization method for glomerular slit membranes in paraffin specimens. The invention carries out dewaxing, hydration, antigen restoration and sealing treatment on the kidney biopsy tissue paraffin section, then sequentially carries out immune marking treatment by using a primary antibody and a secondary antibody, then carries out sealing, and can finish kidney glomerular slit membrane immunofluorescence staining, and then carries out imaging by a super-resolution laser confocal microscope. By using the method of the invention to dye and image the glomerular slit membrane, the three-dimensional structure of the glomerular slit membrane, including the cross-sectional structure and the continuous structure, can be visualized. Changes in the morphology of the slit membrane in the disease state and in the relaxed state can be observed by confocal images, providing direct evidence for assessing the function of the glomerular filtration barrier and exploring the characteristic changes in the slit membrane during remodeling of the filtration barrier.
Description
Technical Field
The invention relates to the technical field of tissue fluorescent staining, in particular to a three-dimensional visualization method for glomerular slit membranes in paraffin specimens.
Background
The glomerular fissure membrane (SLIT DIAPHRAGM) is a special structure on glomerular capillary epithelial cells and has important physiological functions. It is located between the projections of capillary epithelial cells, in the form of a zipper, forming the last layer of filtration barrier, playing a vital role in the filtration function and selective permeability of the glomeruli. First, it allows the water and solutes in the blood to be filtered into the glomeruli and become part of the raw urine. The selective permeability enables the slit membrane to effectively prevent substances such as macromolecular proteins from passing through, thereby maintaining the osmotic pressure and ion balance of body fluid. Second, the glomerular fissure membrane also plays an important role in regulating glomerular filtration rate and hemodynamics. By adjusting the permeability and surface charge of the slit membrane, the kidney can dynamically adjust the filtration rate according to the needs of the organism so as to maintain the stable concentration of various components in blood. In addition, glomerular fissure membranes are involved in the development and progression of diseases such as glomerulonephritis and the like. In the disease state, the structure and function of the slit membrane may be damaged, resulting in the appearance of symptoms such as proteinuria, hematuria, and the like.
Glomerular fissure membranes are composed of a variety of proteins including nephrin and podocin. Nephrin is an important transmembrane protein, mainly present on the cell membrane of podocytes (podocyte) of the glomerulus. It combines with adjacent podocytes through its extracellular structure, forming an important component of the glomerular fissure membrane. The presence of Nephrin helps to maintain the structural integrity of the slit membrane while also participating in signaling and intercellular adhesion, which is critical to maintaining the normal function of the glomerular filtration barrier. On the other hand, podocin is a protein that interacts with nephrin, mainly on the cell membrane of podocytes. Podocin binds to nephrin to form an important protein complex that is critical to maintaining the structure and function of the glomerular filtration barrier. Overall, nephrin and podocin maintain the structural integrity and functional stability of the glomerular filtration barrier as an important component of the glomerular fissure membrane. Their abnormal expression or defective function may lead to the occurrence and development of glomerular diseases, and thus have great significance for normal physiological functions of glomeruli.
The width of the glomerulus fissure membrane is very tiny, usually in the range of 20-50 nanometers, and the glomerulus fissure membrane is observed to be a zipper-like continuous structure through a scanning electron microscope, but only a punctiform structure of a cross section can be observed through a clinical transmission electron microscope (the thickness is 50-70 nanometers), and the continuous form of the glomerulus fissure membrane cannot be seen; in addition, the structure of the slit membrane is tiny, the observation by a transmission electron microscope depends on the dyeing of osmium acid, and if the dyeing is lighter, the slit membrane is not easy to identify. Thus, although electron microscopy is a technique commonly used in clinic to view glomerular ultrastructures, there are the following limitations and disadvantages to the visualization of the slit membrane structure by it: 1. limitation of two-dimensional structure: the ultrathin section used for observation after electron microscope sample preparation is about 70nm, is limited by the angle of the section, and can only observe the cross section structure of the slit film, while the three-dimensional structure of the slit film is complex and continuous, like a zipper-like structure, and cannot be completely displayed in an ultrathin section image; 2. sample restriction: the method is limited by an electron microscope specimen, and an electron microscope ultrathin section usually only has 1-2 glomeruli, and 15-25 glomeruli are used for paraffin kidney biopsy tissue, so that the damage condition of a filter membrane cannot be comprehensively estimated; 3. challenges of sample processing: the steps of fixing an electron microscope sample, preparing a slice, dyeing and the like require high technical skills and complicated operations, and improper sample treatment such as undersize dyeing leads to insufficient dyeing of a slit membrane to influence the observation of a pathologist on the slit membrane; 4. clinical assessment was inadequate: because the slit membrane shown in the electron microscopy image is a cross-sectional structure, it is not possible to provide much information to a clinical pathologist in assessing the damage to the filter membrane in proteinuria patients, resulting in few observation subjects of the ultrastructure of current kidney biopsy puncture specimens involving the slit membrane.
Disclosure of Invention
In order to solve the problems, the invention provides a three-dimensional visualization method for glomerular slit membranes in paraffin specimens. The method provided by the invention can directly realize the observation of the morphology of the glomerular slit membrane of the kidney biopsy paraffin specimen under a confocal microscope after thermal repair treatment and dyeing, and comprises the observation of the area of the glomerulus and/or the length of the glomerular slit membrane.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a method for assisting in observing the morphology of a glomerular fissure membrane, which comprises the following steps:
Using thermal repair to treat the kidney tissue paraffin section, and sealing to obtain a sealed tissue section; the time for thermal restoration treatment of the tissue slices is 9-11 min, and the reagent for thermal restoration treatment of the tissue slices comprises EDTA;
incubating the blocked tissue sections with NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody in sequence, and sealing to obtain the sealed tissue sections;
placing the tissue slice of the sealing slice under a confocal microscope to observe the morphology of the glomerular fissure membrane; the glomerular slit membrane morphology includes an area of glomeruli and/or a length of glomerular slit membrane.
Preferably, the NEPHRIN antibody is available from proteintech under the designation 66970-1-Ig.
Preferably, the thermal repair process includes: and (3) placing the kidney tissue paraffin section into a reagent for thermal restoration treatment of the tissue section, and heating the kidney tissue paraffin section in a pressure cooker for 9-11 min by water.
Preferably, the NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody are diluted in PBS.
Preferably, the NEPHRIN antibody is diluted in a ratio of 1: 600-1: 1000;
the dilution ratio of the ALEXA FLUOR 594-goat anti-mouse antibody is 1: 200-1: 400.
Preferably, the blocking reagent comprises goat serum with the volume concentration of 10%, and the blocking time is 50-60 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method for staining the glomerular slit membrane has better antibody sensitivity and specificity, and can mark all glomerular slit membranes, thereby realizing the observation of a three-dimensional structure, including not only a cross section punctiform structure observed by a clinical transmission electron microscope specimen, but also a continuous structure observed by a scanning electron microscope in scientific research.
(2) Compared with the electron microscope specimen which needs extra tissues and samples, the method does not need extra specimens, can directly use the conventional paraffin embedded tissue section, uses indirect immunofluorescence to complete staining, and saves specimen drawing and sample preparation time.
(3) The method disclosed by the invention is simple in immunostaining process, is a common indirect immunostaining technology, and is low in requirement on technicians and high in feasibility.
(4) After the glomerulus slit membrane is dyed by the method provided by the invention, the renal specimen can be maximally utilized, and the damage condition of the glomerulus slit membrane can be comprehensively evaluated by incorporating all glomeruli.
(5) When the tissue slice dyed by the method provided by the invention is imaged, a confocal microscope is used to obtain enough resolution, so that the efficiency is improved, and the cost is saved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a comparison of different antigen retrieval methods;
FIG. 2 is a graph comparing the repair times of different high pressure thermal antigens;
FIG. 3 is a graph comparing the results of fluorescent staining of primary antibody products;
FIG. 4 is a comparison of imaging effects from different tissue fixation methods and different imaging devices;
FIG. 5 is a graph showing the contrast of an imaging image and an electron microscope image of a healthy glomerular fissure membrane of a paraneoplastic kidney tissue, respectively, using the staining method of the present invention;
FIG. 6 is a graph showing the comparison of an imaging image and an electron microscope image of a glomerular slit membrane of a patient suffering from a remission stage of a micro-lesion nephropathy, and a graph showing measurement of the glomerular sphere area and slit membrane length;
FIG. 7 is a graph showing the comparison of an image of glomerular slit membranes of a patient with a micro-lesion nephropathy with an electron microscope image, respectively, using the staining method of the present invention.
Detailed Description
The invention provides a method for assisting in observing the morphology of a glomerular fissure membrane, which comprises the following steps:
Using thermal repair to treat the kidney tissue paraffin section, and sealing to obtain a sealed tissue section; the time for thermal restoration treatment of the tissue slices is 9-11 min, and the reagent for thermal restoration treatment of the tissue slices comprises EDTA;
incubating the blocked tissue sections with NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody in sequence, and sealing to obtain the sealed tissue sections;
placing the tissue slice of the sealing slice under a confocal microscope to observe the morphology of the glomerular fissure membrane; the glomerular slit membrane morphology includes an area of glomeruli and/or a length of glomerular slit membrane.
The present invention preferably dewaxes and hydrates paraffin sections of kidney tissue. The kidney tissue paraffin section according to the present invention is preferably obtained by a conventional paraffin section preparation method, and the thickness of the kidney tissue paraffin section is preferably the thickness of a clinically conventional sample, more preferably 3 μm. The invention selects the slice thickness of the clinical routine sample, which not only saves sample drawing and sample preparing time, but also has good effect on antibody sensibility and immunostaining resolution of the slice with micron-sized thickness.
In the present invention, the dewaxing preferably comprises the steps of: primarily soaking the kidney tissue paraffin section by using dimethylbenzene; soaking the primarily soaked kidney tissue paraffin section for 30-50 min by using new dimethylbenzene; the primary soaking time is preferably 30-50 min, more preferably 40min; the time for re-soaking is preferably 30-50 min, more preferably 40min. The dewaxing method can completely dewax paraffin sections, avoids the antigen from being not exposed caused by insufficient dewaxing, and provides a good basis for subsequent dyeing.
In the invention, the hydration treatment method comprises the following steps: the dewaxed tissue slice is subjected to a first treatment for 10min by using alcohol with the volume concentration of 100%, the first treated tissue slice is subjected to a second treatment for 10min by using alcohol with the volume concentration of 100%, the second treated tissue slice is subjected to a third treatment for 5min by using alcohol with the volume concentration of 95%, the third treated tissue slice is subjected to a fourth treatment for 5min by using alcohol with the volume concentration of 95%, and the fourth treated tissue slice is subjected to a fifth treatment for 2min by using alcohol with the volume concentration of 80%. The hydration method can better reduce tissues to an in-vivo state, and avoid the problems of uneven dyeing, non-specific background coloring and the like.
After the tissue slice is hydrated, the invention uses thermal repair to treat the kidney tissue paraffin slice and seals the kidney tissue paraffin slice to obtain a sealed tissue slice. In the invention, the time for thermal repair treatment of the tissue slices is 9-11 min, preferably 10min; the thermal repair process preferably includes: placing the kidney tissue paraffin section into a reagent for heat repairing treatment of the tissue section, and heating the kidney tissue paraffin section by water in an autoclave; the pressure of the thermal repair treatment tissue slice is preferably 80-100 kilopascals; the agent for thermal restoration treatment of tissue slices comprises EDTA; the pH of the EDTA is preferably 9.0; the temperature of the enclosure is preferably room temperature; the blocking reagent preferably comprises goat serum with the volume concentration of 10%, and the blocking time is preferably 50-60 min, more preferably 55min. In the thermal repair treatment time, the morphology of the glomerular fissure membrane cannot be displayed when the treatment time is too short; the treatment time is too long and nonspecific staining occurs. According to the invention, through proper thermal repair treatment time, only one antigen repair is carried out, so that antigens can be completely exposed, and non-specific staining can not occur.
After obtaining the tissue slice subjected to the sealing treatment, the invention sequentially incubates the tissue slice subjected to the sealing treatment with NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody, and seals the tissue slice to obtain the tissue slice of the sealing piece. In the present invention, the NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody are preferably used after being diluted with PBS solution; the NEPHRIN antibody is preferably diluted in a ratio of 1: 600-1: 1000, further preferably 1: 800-1: 900, more preferably 1:800; the NEPHRIN antibody is used for incubating the tissue slice subjected to the sealing treatment preferably at the temperature of 4 ℃, and the incubation time is preferably 12-14 h, more preferably 12h; the dilution ratio of the ALEXA FLUOR 594-goat anti-mouse antibody is preferably 1: 200-1: 400, more preferably 1:300; the ALEXA FLUOR 594-goat anti-mouse antibody is preferably used for incubating the tissue section incubated by the NEPHRIN antibody for 0.5-1 h at room temperature, and the incubation time is more preferably 1h. In a specific embodiment of the invention, it is preferred that after treatment of the tissue sections with NEPHRIN antibody, the tissue sections are washed 3 times with PBS buffer for 3min each time, and then treated with ALEXA FLUOR 594-goat anti-mouse antibody. In the present invention, the NEPHRIN is preferably available from proteintech under the trade designation 66970-1-Ig. The NEPHRIN antibody selected by the invention can be used as a primary antibody for marking tissue sections, so that glomerular fissure membranes in normal states and all disease states can be stained and visualized.
In the present invention, the caplet used in the caplet is preferably DAPI (cat#d5942 Sigma). Before sealing, the tissue slice is preferably washed for 3 times with PBS buffer solution for 3min each time, so that the nonspecific adsorption secondary antibody is guaranteed to be cleaned, the nonspecific color development is reduced, the background is guaranteed to be clean, and the tissue slice is preserved in a dark place after sealing.
After obtaining the tissue slice of the sealing slice, the invention places the tissue slice of the sealing slice under a confocal microscope to observe the morphology of the glomerular fissure membrane; the glomerular slit membrane morphology includes an area of glomeruli and/or a length of glomerular slit membrane.
In the method for observing the morphology of the glomerular slit membrane, the confocal microscope is preferably used for imaging, the super-resolution confocal imaging is preferred for the adopted imaging mode, the device model is preferably nikon AX R WITH NSPARC, the image acquisition condition is preferably that a 100×oil immersion objective NA1.4 is used, the acquisition size is preferably 163×163 μm, the image with the single-layer thickness of preferably 0.3 μm is set, continuous shooting is preferred, and the shooting layer number is preferably 10.
In the invention, the photographed image is preferably subjected to image post-processing by using image processing software, wherein the image processing software is preferably NIS-ELEMENTS AR, and the specific operation is preferably using Lucy-Richardson deconvolution, and the most preferably using 5 times.
In the invention, the deconvoluted image is preferably subjected to maximum projection, and the obtained image can obtain a three-dimensional image of an in-situ slit membrane structure of the kidney biopsy puncture paraffin tissue.
In a specific embodiment of the invention, morphological features of the glomerular fissure membrane in the three-dimensional image are observed, including but not limited to the following morphological parameters: 1) Similarity between continuity and integrity of glomerular fissure membranes and morphology; 2) Length of glomerular fissure membrane per unit area. In a specific embodiment of the present invention, the measurement method preferably uses an ImageJ image processing software system to perform the measurement of the glomerular slit membrane morphological parameters.
The measurement method for the glomerular fissure membrane length per unit area in the three-dimensional image is preferably as follows: opening the picture by using ImageJ software to perform circling on the glomerular target area (Polygon selections); measuring the area of the selected region (analysis-measure), scoring (SEGMENTED LINE) along the glomerular fissure membrane, and measuring the segment (analysis-measure); the ratio of the obtained length to the area can reflect the damage degree of the glomerular fissure membrane. A smaller ratio of length/area represents more severe damage.
The immunofluorescence staining method provided by the invention can be used for visualizing the three-dimensional structure of the glomerular slit membrane of the paraffin specimen by combining confocal microscopy imaging, and comprises a cross-sectional structure and a continuous structure. Changes in the morphology of the slit membrane in the disease state and in the relaxed state can be observed by confocal images.
For further explanation of the present invention, the following describes in detail a method for visualizing the glomerular fissure membrane in paraffin specimens provided by the present invention, with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of protection of the present invention.
Example 1
A method for immunofluorescence staining of glomerular fissure membranes, which comprises the following steps:
1. treatment prior to immunolabeling of paraffin sections of kidney biopsies:
1) Kidney biopsy tissue paraffin section: patient kidney biopsies were fixed using conventional 4.5% formalin and sectioned to a thickness of 3 μm after conventional paraffin embedding;
2) Dewaxing and hydrating: paraffin sections were sequentially passed through xylene and alcohol cylinders: xylene 40min,100% alcohol 10min,95% alcohol 5min,80% alcohol 2min, and then PBS washing 3 times for 3min each;
3) Antigen retrieval: the antigen repairing method is EDTA high-pressure repairing, and the specific method comprises the following steps: heating and boiling the autoclave, and then placing paraffin tissue slices into EDTA repair liquid (pH value is 9.0) of the autoclave, and heating the paraffin tissue slices to boiling; covering a pressure valve until the steam is sprayed for 10min;
4) Closing: blocking 10% goat serum at room temperature for 30min;
2. Performing immune labeling treatment on paraffin section glomerular slit membranes:
1) An anti-treatment: dilution ratio 1 with PBS: 800 dilution of murine anti-human NEPHRIN antibody (designated antibody 3), incubation of tissue sections overnight at 4 ℃ using diluted antibody;
2) Secondary antibody treatment: tissue sections were washed 3 times with PBS 3min each, diluted 1 with PBS: 300 dilution of ALEXA FLUOR 594-goat anti-mouse antibody, incubation of tissue sections with diluted antibody at room temperature for 1h;
3) DAPI-encapsulation tablet encapsulation: the tissue sections were washed 3 times with PBS for 3min each, blocked (chemically stained) with DAPI-containing blocking agents, and stored protected from light after blocking.
Example 2
Unlike example 1, the antigen retrieval time was 9min, with the remaining steps unchanged.
Example 3
Unlike example 1, the antigen retrieval time was 11min, with the remaining steps unchanged.
Comparative example 1
The subsequent experimental procedure of example 1 was performed using kidney tissue frozen section 1; the preparation process of the frozen section 1 is as follows: the kidney puncture biopsy tissue is placed into an embedding mould with the OCT, precooled OCT is continuously added into the embedding mould, the tissue is completely covered, and the tissue is sliced in a frozen microtome, wherein the thickness of the tissue is about 5 mu m. Frozen section 1 was washed 3 times in PBS for 3min each, then sheep serum was blocked, and primary and secondary antibodies, isoparaffin tissue were added.
Comparative example 2
Unlike example 1, the antigen retrieval method was microwave EDTA retrieval, the remaining steps were unchanged; the microwave EDTA repairing method comprises the following steps: heating to boiling with 100W of microwave fire, taking out the beaker, placing the tissue slice into boiling EDTA repair liquid (pH value is 9.0), placing into the microwave oven again, reducing fire to 40W, heating for 10min, and naturally cooling.
Comparative example 3
Unlike example 1, the antigen retrieval method was citric acid high pressure retrieval, the remaining steps were unchanged; the method for repairing the citric acid under high pressure comprises the following steps: placing paraffin tissue slices into a citric acid restoration solution (pH value is 6.0) of the autoclave, and heating to boil; the pressure valve is covered until the vapor is sprayed for 5min.
Comparative example 4
Unlike example 1, the antigen retrieval was performed using 0.4% pepsin, specifically 100. Mu.l of 0.4% pepsin digest (ZLI-9013) was added dropwise to the tissue sections, the volume of addition was such that the tissue could be covered, and incubation was carried out for 30min at 37℃with the remaining steps unchanged.
Comparative example 5
Unlike example 1, the time for antigen retrieval was 5min, with the remaining steps unchanged.
Comparative example 6
Unlike example 1, the time for antigen retrieval was 15min, the remaining steps were unchanged.
Comparative example 7
Unlike example 1, the rabbit anti-human NEPHRIN antibody was replaced with antibody 1 at the time of primary antibody treatment, and antibody 1 was purchased from OriGene Technologie company under the accession number BP5030, with the corresponding secondary antibody, and the remaining steps were unchanged.
Comparative example 8
Unlike example 1, the rabbit anti-human NEPHRIN antibody was replaced with antibody 2 at the time of primary antibody treatment, and antibody 2 was purchased from Merck company under the designation PRS2265, with the corresponding secondary antibody, and the remaining steps were unchanged.
Comparative example 9
Unlike example 1, the imaging apparatus was a general fluorescence microscope.
Test example 1
Placing the fluorescence-dyed sections of the example 1 and the comparative examples 1-8 under a 100-time common fluorescence microscope, and integrally evaluating the immunostaining condition of glomerular slit membranes to ensure that all the slit membranes of the glomerulus are fluorescence-positive; the slice is placed under a 100-fold confocal fluorescence microscope (meanwhile, comparative example 9) for shooting imaging, deconvolution treatment is carried out, and morphological characteristics of the glomerular slit membrane are observed and measured. The results are shown in fig. 1-4.
FIG. 1 is an image of paraneoplastic kidney tissue imaged with a 100-fold super-resolution confocal microscope using different antigen retrieval methods, wherein the scale of the left panel is 50 μm; the scale of the right detail drawing is 5 μm. As can be seen from fig. 1, the microwave high temperature EDTA repair (a) in fig. 1, the gastric enzyme repair (b) in fig. 1, the citric acid repair (c) in fig. 1), the slit membrane (SD) was stained positive, but the antigen was not fully exposed, the antigen was exposed by about 50% -70%, and in addition, the glomerular slit membrane structure was not etched out and could not be distinguished; example 1 of the present invention uses high pressure EDTA thermal repair (d in fig. 1) with 100% antigen exposure, the glomerular slit membrane structure was etched out easily discernable.
FIG. 2 is a comparison of paraneoplastic kidney tissue at various times during the autoclave repair, wherein the scale on the left panel is 5 μm; the scale of the right detail drawing is 1 μm. As can be seen from fig. 2, EDTA autoclave antigen repair for 5min (a in fig. 2), glomeruli were positive, partial slit membrane (SD) antigen exposure, about 40% of antigen exposure, but glomerular slit membrane structure was indistinguishable; EDTA autoclave antigen repair for 15min (b in FIG. 2), glomeruli were positive, all slit membrane antigens were exposed, but there was nonspecific staining, resulting in a blurred and indistinguishable glomerular slit membrane structure; example 1 of the present invention was heat-repaired using high pressure EDTA for 10min (c in fig. 2), with 100% antigen exposure, the glomerular fissure membrane structure was completely etched out, and the punctate structure (SD-a) and the continuous serpentine structure (SD-b) were clearly distinguished.
FIG. 3 is a comparative graph of immunolabeling of paraneoplastic kidney tissue using different antibody products, with the left large scale of 10 μm and the right detailed scale of 2 μm. As can be seen from fig. 3, the staining of antibody 1 (a in fig. 3) marks glomerular fissure membrane of glomeruli to some extent, but the fluorescence signal intensity is weak, and glomerular fissure membrane Structure (SD) cannot be completely etched; antibody 2 (b in fig. 3) can mark most of the slit membranes, the antigen marks about 80%, the specificity to the glomerular slit membranes is strong, but the slit membrane has poor structural continuity, only punctiform structures (SD-a) can be etched, and serpentine structures can not be etched; the NEPHRIN antibody (antibody 3, c in fig. 3) used in the present invention has good specificity and sensitivity to glomerular slit membrane markers, and the signals can be seen to be distributed along the capillary wall, and the punctiform morphology (SD-a) and the serpentine morphology (SD-b) of the glomerular slit membrane can be distinguished from each other by the signals.
Fig. 4 is a comparison of paraneoplastic kidney tissue using different tissue fixation methods and different imaging modalities. As can be seen from fig. 4, using frozen tissue (a in fig. 4), glomerular markers were positive, but slit membrane Structure (SD) morphology was poorly preserved; imaging paraffin tissue (b in fig. 4) after nephrin immunolabeling under a common fluorescence microscope can show the morphology of the slit membrane, but the outline is not clear enough; after imaging by a super-resolution confocal microscope (c in fig. 4), the complete slit membrane punctiform structure (SD-a) and serpentine structure (SD-b) can be displayed. The slit membrane structure observed by the transmission electron microscope (d in fig. 4) is located between adjacent foot processes, and is limited by the sample preparation and imaging modes, and one view can only display the slit membrane of the local capillary vessel wall.
Example 4
The renal small sphere area and the slit membrane length obtained in example 1 were measured in a specific manner comprising the steps of:
Picture was opened using ImageJ software to delineate the area of glomeruli (d in fig. 6), toolbar-polygon selections, measure the area of the circled site, analyze-measure; the area of glomeruli can be obtained. Measuring the length of the slit film, firstly converting an image from RGB to a gray image (g in fig. 6), namely image-type-8-bit, then identifying the slit film through Plugins-edge Detection plug-in (h in fig. 6), and clicking the measurement to obtain the length of the slit film.
Test example 2
The image of immunostaining and the electron microscope image of the ultrathin section of the healthy glomerular slit membrane obtained from the paraneoplastic renal tissue were respectively subjected to the method of example 1, and the morphology of the glomerular slit membrane was observed and the glomerular slit membrane length/area ratio was measured. The result is shown in FIG. 5, where b is a detailed view within the box in a.
As can be seen from fig. 5, the whole glomeruli can be displayed using a super-resolution confocal microscope, with an image size of 163 μm (long) ×163 μm (wide) ×2 μm (thick) of the slit membrane (a in fig. 5), and thus the 3D structure of the slit membrane can be displayed including not only the dot-like structure (SD-a, f in fig. 5, and g in fig. 5) visible in the electron microscope image, but also the continuous linear serpentine structure (SD-b, D in fig. 5); transmission electron microscopy (c in fig. 5), with an image size of 18 μm (length) x 14 μm (width) x 70nm (thickness), a slit membrane Structure (SD) was located between adjacent foot processes (f in fig. 5 and g in fig. 5), limited to the manner of sample preparation and imaging, and only slit membranes of local capillary walls were displayed. The slit film length/area ratio was 0.61 μm -1.
Test example 3
Kidney biopsies from patients with minimal lesion kidney disease in remission were immunostained and transmission electron microscopy-sampled electron microscopy pictures of ultrathin sections, respectively, by the method of example 1, and the morphology of glomerular slit membranes was observed and the glomerular slit membrane length/area ratio was measured. The result is shown in fig. 6, where b is a detailed view within the box in a and f is an enlarged view within the box in c.
As can be seen from fig. 6, the whole glomeruli (a in fig. 6) can be displayed under a super-resolution confocal microscope at 100 times, the continuous morphology of the slit membrane (SD-b, e in fig. 6) can be seen in an enlarged view, and the electron microscopy image is limited to the slice thickness, i.e., only 70nm, and thus the cross section of the continuous morphology (f in fig. 6) can be seen even. The slit film length/area ratio was 0.54 μm -1.
Test example 4
Kidney biopsies from patients with micro-lesions in the period of kidney disease were immunostained and transmission electron microscopy-sampled in an electron microscope image of ultrathin sections, respectively, by the method of example 1, and the morphology of glomerular slit membrane was observed and the glomerular slit membrane length/area ratio was measured. The result is shown in fig. 7, where b is a detailed view within the box in a and f is an enlarged view within the box in c.
As can be seen from fig. 7, the entire glomeruli (a in fig. 7) can be visualized using a super-resolution confocal microscope at 100 x, and the enlarged view can reveal various morphologies of the slit membrane, including the punctiform structures under cross-section (SD-a, d in fig. 7), and different continuous serpentine morphologies (SD-b, e in fig. 7 and g in fig. 7), which are different from paraneoplastic kidney tissue (d in fig. 5) and remission tissue (e in fig. 6). Only the punctiform morphology of the slit film (f in FIG. 7) can be seen in the electron microscopy image. The slit film length/area ratio was 0.44 μm -1.
In conclusion, compared with the method, the glomerular slit membrane is dyed, and the 3D structure of the glomerular slit membrane, including punctate morphology and continuous structure, can be clearly displayed. The morphology of the slit membrane in proteinuria disease presents a different morphology; whereas after treatment, the slit membrane has been reconstructed and the morphology tends to paraneoplastic kidney tissue. On one hand, the traditional concept and the technical barrier that the morphology of the glomerular fissure membrane can only be observed under an electron microscope are broken; on the other hand, the structure of glomerular fissure membranes of all kidney biopsy puncture tissues can be examined, so that a pathologist can evaluate the damage of the filter membranes more comprehensively; in addition, the method is a common indirect immunostaining technology, is simple to operate, has low requirements on technicians, and has high feasibility.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (4)
1. A method for assisting in observing the morphology of glomerular fissure membranes, which is characterized by comprising the following steps:
Dewaxing and hydrating the kidney tissue paraffin section, then using thermal repairing to treat the kidney tissue paraffin section, and sealing to obtain a sealed tissue section; the method for treating the paraffin sections of the kidney tissues by thermal repair is EDTA high-pressure repair; the time for thermal repair treatment of the kidney tissue slices is 9-11 min, and the reagent for thermal repair treatment of the kidney tissue slices is EDTA;
Incubating the blocked tissue sections with NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody in sequence, and sealing to obtain the sealed tissue sections; the NEPHRIN antibody is purchased from proteintech company under the product number 66970-1-Ig;
Placing the tissue slice of the sealing slice under a confocal microscope to observe the morphology of the glomerular fissure membrane;
The method for observing the morphology of the glomerular fissure membrane comprises the following steps: shooting and imaging by using the confocal microscope, and then performing deconvolution treatment to obtain a deconvoluted image; performing maximum projection on the deconvoluted image to obtain a three-dimensional image of an in-situ slit membrane structure of renal paraffin tissue;
the confocal microscope is nikon AX R WITH NSPARC super-resolution confocal microscope;
The glomerular slit membrane morphology includes the area of glomeruli and the length of glomerular slit membrane.
2. The method of claim 1, wherein the NEPHRIN antibody and ALEXA FLUOR 594-goat anti-mouse antibody are used after dilution with PBS solution.
3. The method of claim 2, wherein the NEPHRIN antibody is diluted in a ratio of 1: 600-1: 1000;
the dilution ratio of the ALEXA FLUOR 594-goat anti-mouse antibody is 1: 200-1: 400.
4. The method of claim 1, wherein the blocking reagent comprises goat serum at a concentration of 10% by volume for 50-60 min.
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