CN113984483A - Staining method for frozen section cover glass paster - Google Patents
Staining method for frozen section cover glass paster Download PDFInfo
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- CN113984483A CN113984483A CN202111145297.7A CN202111145297A CN113984483A CN 113984483 A CN113984483 A CN 113984483A CN 202111145297 A CN202111145297 A CN 202111145297A CN 113984483 A CN113984483 A CN 113984483A
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- 238000007447 staining method Methods 0.000 title claims abstract description 7
- 241001272567 Hominoidea Species 0.000 claims abstract description 11
- 239000011148 porous material Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 32
- 239000008055 phosphate buffer solution Substances 0.000 claims description 16
- 238000010186 staining Methods 0.000 claims description 16
- 238000011534 incubation Methods 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 230000000903 blocking effect Effects 0.000 claims description 9
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000007605 air drying Methods 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 239000012224 working solution Substances 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 4
- 239000003517 fume Substances 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
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- 238000002474 experimental method Methods 0.000 description 7
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- 238000007667 floating Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- 230000002055 immunohistochemical effect Effects 0.000 description 5
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
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- 210000004556 brain Anatomy 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention discloses a staining method for frozen section cover glass patches, which directly pastes tissues after frozen section on a cover glass treated by APES and carries out subsequent immunostaining steps in a pore plate. Not only the operation steps become simple and easy, but also the times of repeated use of the primary resist are greatly improved, the cost is effectively saved, and meanwhile, high-quality pictures can be obtained.
Description
Technical Field
The invention belongs to the technical field of pathological sections, and particularly relates to a staining method for a frozen section cover glass paster.
Background
Frozen section immunostaining is a technique of in situ tissue detection of the distribution of antigens (or antibodies) in tissues using labeled specific antibodies (or antigens). The main steps of the immunostaining experiment of the frozen section comprise tissue sampling, fixing, dehydrating, embedding, slicing, picking, pasting, antigen repairing (optional steps), sealing, antibody incubation, mounting, microscopic imaging and the like. The traditional immunostaining of frozen sections includes a patch method and a floating slice method.
The patch method is most commonly used for paraffin sectioning, but can also be used for frozen sectioning. The slide used in the patch method requires pretreatment in advance. After the tissue was sectioned by a freezing microtome, it was immediately mounted on a sheet, air-dried, and then stored in a refrigerator at-20 ℃ for later use. Before the method is used for sealing, an immunohistochemical pen is used for defining a range around tissues to prevent sealing liquid or antibody from diffusing to influence dyeing. The volume of the antibody required by the patch method is small, the antibody can not be recovered, the integrity of the tissue can be well maintained, but when the antibody is eluted, the whole glass slide needs to be soaked in a detergent, so that the residual antibody which is not combined can be well removed, and the background is reduced. In addition, the disadvantages of the patch method include: (1) although immunohistochemical pen circles may prevent antibody diffusion to some extent, it is difficult to avoid antibody diffusion or volatilization during overnight incubation of the primary antibody, resulting in poor staining. (2) Slide detachment can easily occur if the slide is improperly pretreated or if the tissue does not dry well on the slide. (3) Antibody incubation temperatures and punch times need to be explored multiple times. (4) The primary antibody and the secondary antibody need to be washed in a staining jar after finishing, and more reagents are consumed.
The floating method is that the tissue sample is in a floating state before mounting, and is not mounted on a glass slide or a cover glass. After the tissue is frozen and sliced, the slices are directly stored in a freezing-free solution (the ratio of glycerol to ethylene glycol to PBS buffer solution is 3: 3: 4) at the temperature of-20 ℃ for later use. Prior to staining, tissue sections were transferred to PBS buffer in 24 or 12 well plates, washed three times and subjected to subsequent sealing and staining steps. After antibody incubation and rinsing, the tissue was transferred to a slide for mounting. The antibody can enter the tissue better in the bleaching method, and the dyeing effect is better. However, the disadvantages of the floating sheet method include: (1) special requirements are provided for slice thickness and slice preservation mode. (2) After more than ten operations, the film is easy to rot, and the final sealing of the fragments is difficult. (3) Antibody staining and uneven rinsing are easily caused, and (4) when the antigen is repaired, tissue is easily curled or broken when the tissue piece is directly boiled in a repairing liquid.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a staining method for a frozen section cover glass patch.
The technical scheme of the invention is as follows:
a staining method for a frozen section cover glass patch comprises the following steps:
(1) soaking the cleaned cover glass with fresh APES working solution, cleaning with distilled water to remove unbound APES, and air drying to obtain pretreated cover glass;
(2) placing the pretreated cover fragments into corresponding holes of a multi-hole plate, immersing the pretreated cover fragments in PBS (phosphate buffer solution), transferring the frozen sections into the corresponding holes by using a soft writing brush, slightly pressing the frozen sections by using the writing brush to enable the frozen sections to be sunk on the pretreated cover glass, and enabling the cover glass to be flatly laid at the bottoms of the corresponding holes by the sizes of the corresponding holes;
(3) removing the PBS while preventing the frozen section from being sucked out, and adjusting the position of the frozen section on the pretreatment cover glass with the aid of a writing brush;
(4) drying the frozen section tissue in the pore plate until the tissue turns white from transparent, adding PBST buffer solution, rinsing on a decoloring shaker at room temperature for 5min, adding confining liquid, and incubating at room temperature for 1 h;
(5) removing the blocking solution, adding primary antibody solution, and incubating at 2-4 deg.C for 12-16 h;
(6) after the incubation is finished, recovering the primary antibody solution, rinsing the primary antibody solution on a decoloring shaker at room temperature for 3 times, 10-15min each time, and then adding a fluorescent secondary antibody solution containing DAPI to incubate for 1h at room temperature in a dark place;
(7) after incubation, the fluorescent secondary antibody solution is removed, PBST is rinsed 3 times in a dark place for 10-15min each time, and finally, mounting is carried out.
In a preferred embodiment of the present invention, in the step (1), the washed cover glass is placed in a glass container and soaked with fresh APES working solution.
In a preferred embodiment of the present invention, the thickness of the cryosection may be in the range of 40 μm.
In a preferred embodiment of the present invention, the drying in the step (4) is room temperature air drying or oven drying.
Further preferably, the room temperature air drying is carried out in a fume hood for 20-30 min.
Further preferably, the drying temperature of the oven is 37 ℃ and the drying time is 10 min.
In a preferred embodiment of the present invention, the solvent of the primary antibody solution is the blocking solution.
More preferably, 0.01% sodium azide is added to the primary antibody solution.
In a preferred embodiment of the present invention, the solvent of the fluorescent secondary antibody solution is the blocking solution.
In a preferred embodiment of the invention, the mounting is washed with PBS prior to mounting.
The invention has the beneficial effects that:
1. the tissue after frozen section is directly pasted on a cover glass processed by APES, and the subsequent immunostaining step is carried out in a pore plate. Not only the operation steps become simple and easy, but also the times of repeated use of the primary resist are greatly improved, the cost is effectively saved, and meanwhile, high-quality pictures can be obtained.
2. The antibody in the invention is very convenient to recover, and the repeated use times and time of the antibody are prolonged. The subsequent elution does not combine the liquid of the first antibody and the second antibody, and the good elution effect can be achieved by using less liquid.
3. The method is simple to operate, the whole subsequent process is carried out in the pore plate, no matter the antibody is incubated or rinsed, the tissue slice does not need to be transferred, and the possibility of sample damage is greatly reduced.
4. The invention has good paster effect and the tissue is not easy to dry, the traditional paster dyeing antibody is dripped in the circle of the immunohistochemical stroke, and the sample dyeing failure caused by antibody diffusion is difficult to avoid, and the invention can well avoid the problem.
5. The invention can simultaneously carry out large-batch experiments, each cover glass can be used for holding 2-5 frozen section samples according to the size of the tissue, one 12-pore plate can be used for simultaneously carrying out experiments on 12-36 different samples, the repeatability of the samples is good, the occupied area of the experiment operation is small, and the pore plates can be overlapped and placed even in large-batch experiments.
6. The invention needs no additional experimental equipment, and the traditional patch method needs a dark box and an immunohistochemical pen.
7. Although the existing bleaching method has good dyeing effect, after multiple steps, the tissue sample is seriously damaged and even loses the sample during washing; the invention can well keep the morphological structure of the tissue sample and greatly improve the success rate of dyeing.
Drawings
Fig. 1 is a schematic flow chart of embodiment 1 of the present invention.
FIG. 2 is a graph showing the effect of the experiment according to the embodiment of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1
As shown in fig. 1, a method for staining a frozen section coverslip patch includes the following steps:
(1) cover glass pretreatment: taking a 12-well plate as an example, a round cover glass with a diameter of 20mm is placed in a glass dish (taking care to avoid using a disposable plastic culture dish, and the plastic dish is easily corroded by acetone), soaked and treated for 1-2min by using a fresh APES (3-Aminopropyrophyllythoxysilane, 3-triethoxysilyl-1-propylamine) working solution (diluted by 1:50 acetone or according to the corresponding product specification) (the APES working solution contains acetone, the experiment operation needs to be carried out in a fume hood, an organic solvent recovery bottle needs to be poured after the working solution is used for treatment), washed by distilled water for 2-3 times to remove unbound APES, then dried in the fume hood to obtain a pretreated cover glass, and placed at 4 ℃ for later use, and dust is prevented.
(2) Tissue sticking: placing the pretreated cover fragments into corresponding holes of a 12-hole plate, immersing the pretreated cover fragments in about 1mL of PBS buffer solution, transferring the frozen section tissues (in the embodiment, the frozen section of the E16.5 (embryo 16.5d) mouse brain tissues, the thickness of which is 40 mu m) into the corresponding holes by using a soft writing brush, and slightly pressing the frozen section tissues by using the writing brush to make the frozen section bottom on the pretreated cover glass; gently suck the PBS buffer solution in the hole with a pipette while preventing the frozen section tissue from being sucked out, and adjust the position of the frozen section on the pretreatment cover glass with the help of a writing brush. Tissue sections in 12-well plates were dried to turn white from clear.
The drying comprises the following steps: placing in a ventilated kitchen, air drying at room temperature, and taking about 20-30min when the tissue turns white from transparent to dry; or placing in oven at 37 deg.C for quick drying for about 5-10 min; note that: if the tissue is not completely dried, the tissue is prone to flaking and aspiration of the sample during subsequent processing should be avoided.
(3) And (3) sealing: the attached tissue sections were rinsed 1-2 times with PBST (5 min each), followed by incubation for 1h on a 300. mu.L blocking solution incubator with a shaker, and the blocking solution was removed.
(4) Incubating the primary antibody: after diluting the primary antibody with the blocking solution according to the corresponding antibody dilution ratio, the primary antibody is added into a pore plate with a tissue section attached, and incubated overnight (12-16h) at 4 ℃, wherein about 300-350 mu L of the primary antibody solution is needed in each corresponding pore to submerge the cover glass and the tissue sample. The antibody dilution ratio and related information in this example are shown in Table 1.
(5) Elution of unbound primary antibody: after incubation, the primary antibody solution was recovered to 1.5ml EP tubes using a pipette gun. The PBST buffer was rinsed on a decolorizing shaker at room temperature and repeated three times (500. mu.L per well for 10-15min each) to remove unbound primary antibody.
Note that: the recovered primary anti-solution is sealed and stored at 4 ℃, the solution is clear and has no turbidity before the next use, and the effect can be reduced after the solution is used for many times. Through laboratory test statistics, more than 90% of primary antibodies can be repeatedly used for more than 4 times within half a year; the antibody can be stored for more than half a year, and 80% of the antibody can be repeatedly tested for at least 2 times.
(6) Incubation of secondary antibody: the corresponding secondary antibody cross-linked with fluorescent group (such as Alexa 488,568,647, etc.) and DAPI are diluted in the blocking solution according to the appropriate dilution ratio and incubated on a decolorized shaker for 1h at room temperature in the absence of light. The formulation of the secondary antibody in this example was Goat-anti rat Alexa Fluor 4881: 1000, Goat-anti rabbitt Alexa Fluor 5681: 1000, DAPI 1mg/mL from Thermo Fisher company, and the solvent was blocking solution.
(7) Elution of unbound secondary antibody: after the secondary antibody incubation was completed, the secondary antibody was removed (the secondary antibody was not generally reused), and washed three times (15 min each) with PBST buffer in the dark at room temperature to remove unbound secondary antibody;
(8) and (3) mounting and imaging: after the elution of the secondary antibody, PBST was replaced with PBS and washed again to reduce the appearance of bubbles during mounting. The coverslip was removed with ophthalmic forceps, drained and gently applied upside down to the slide with the mounting tablet (containing the anti-quencher) added, indicating the date and sample information. The edges of the coverslips were sealed with nail polish to prevent dropping. And (3) keeping out of the sun, drying at room temperature for 10-20min, imaging the immunostained tissue piece by using a common fluorescence microscope or a confocal microscope, and storing in a slide box at-20 ℃ for subsequent imaging.
PBS buffer formulation: NaCl: 8g of the total weight of the mixture; KCl: 0.2 g; KH (Perkin Elmer)2PO 4：0.27g；Na 2HPO 4·12H 2O: 3.58 g. Dissolved in 1L ddH2O, adjusting pH to 7.4.
PBST buffer formulation: 0.2% TritonX-100 was added to PBS buffer.
The formula of the sealing liquid is as follows: 10% fetal bovine serum (v/v), 5% BSA (m/v), 0.2% TritonX-100, 0.01% NaN3And dissolved in PBS buffer.
TABLE 1 immunostaining antibody information for this example
Compared with the traditional patch method and the traditional floating method (the specific operation is shown in Zhanhe et al, the effect of the floating method and the patch method in the brain tissue section immunohistochemical technology is compared, the academy of New county medical sciences, 2006.23(1): p.9-11.), the method (cover glass patch method) is obviously more convenient and faster in experimental operation. As shown in FIG. 2, the immunofluorescence staining of two proliferation marker antibodies Brdu and ki67 was performed on E16.5 (embryo 16.5d) mouse brain sections by using the three methods at the same time, and the results show that the three methods can obtain better staining effect. Among the three methods, although the background of the bleaching method is low, the time of the acidification treatment process is difficult to control, the brain slices are easy to curl, and the dyeing success rate is low. The method of the present invention shows a lower background signal due to a more thorough step of eluting unbound antibody compared to the patch method.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A staining method for a frozen section cover glass patch is characterized by comprising the following steps: the method comprises the following steps:
(1) soaking the cleaned cover glass with fresh APES working solution, cleaning with distilled water to remove unbound APES, and air drying to obtain pretreated cover glass;
(2) placing the pretreated cover fragments into corresponding holes of a multi-hole plate, immersing the pretreated cover fragments with PBS (phosphate buffer solution), transferring the frozen section tissues into the corresponding holes by using a soft writing brush, slightly pressing the frozen section tissues by using the writing brush to enable the frozen section tissues to sink to the bottom of the pretreated cover glass, and enabling the cover glass to be flatly laid at the bottom of the corresponding holes by the size of the corresponding holes;
(3) removing the PBS, simultaneously avoiding the frozen section tissue from being sucked out, and adjusting the position of the frozen section tissue on the pretreatment cover glass with the aid of a writing brush;
(4) drying the frozen section tissue in the pore plate until the tissue turns white from transparent, adding PBST buffer solution, rinsing on a decoloring shaker at room temperature for 1-2 times, 5min each time, adding confining liquid, and incubating at room temperature for 1 h;
(5) removing the blocking solution, adding primary antibody solution, and incubating at 2-4 deg.C for 12-16 h;
(6) after the incubation is finished, recovering the primary antibody solution, rinsing the primary antibody solution on a decoloring shaker at room temperature for 3 times, 10-15min each time, and then adding a fluorescent secondary antibody solution containing DAPI to incubate for 1h at room temperature in a dark place;
(7) after incubation, the fluorescent secondary antibody solution is removed, PBST is rinsed 3 times in a dark place for 10-15min each time, and finally, mounting is carried out.
2. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: in the step (1), the cleaned cover glass is placed in a glass container and soaked in fresh APES working solution.
3. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: the thickness of the frozen sections is in the range of 20-200 μm.
4. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: and (4) drying in the step (4) is room temperature air drying or oven drying.
5. A method of staining a frozen section coverslip patch as claimed in claim 4 wherein: the room temperature air drying is carried out in a fume hood for 20-30 min.
6. A method of staining a frozen section coverslip patch as claimed in claim 4 wherein: the drying temperature of the oven is 37 ℃ and the drying time is 5-10 min.
7. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: the solvent of the primary anti-solution is the confining liquid.
8. A method of staining a frozen section coverslip patch as claimed in claim 7 wherein: 0.01% of sodium azide is added into the primary antibody solution.
9. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: the solvent of the fluorescent secondary antibody solution is the confining liquid.
10. A method of staining a frozen section coverslip patch as claimed in claim 1 wherein: before the mounting, washing with PBS was performed.
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