CN115855933A - Pig muscle fiber diameter determination method based on Image J Image analysis - Google Patents
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Abstract
The invention relates to the technical field of animal muscle fiber diameter measurement, in particular to a pig muscle fiber diameter measurement method based on Image J Image analysis. The method comprises the following steps: s1, acquiring a pig skeletal muscle fiber image; (1) collecting a pig skeletal muscle sample; preparing a skeletal muscle paraffin section; (3) HE staining of paraffin sections; (4) image acquisition; s2, carrying out quantitative analysis processing on the Image of the S1 by using Image J software; and S3, calculating the diameter of the muscle fiber. The prepared paraffin section of the skeletal muscle is photographed after HE dyeing, and the picture is processed and analyzed by using Image J, so that the method for obtaining the better diameter of the skeletal muscle fiber of the pig is obtained, the influence of the change of the shape of the muscle fiber and the type of the muscle fiber after HE dyeing on the diameter result of the muscle fiber is reduced, the conditions of long time consumption, low measuring efficiency and human errors of the conventional muscle fiber measurement are avoided, the rapid batch measurement can be realized, and the operation is simple.
Description
Technical Field
The invention relates to the technical field of animal muscle fiber diameter measurement, in particular to a pig muscle fiber diameter measurement method based on Image J Image analysis.
Background
With the improvement of the per capita income level and the popularization of the healthy consumption concept in the healthy age, consumers have more preference for safe, high-quality and characteristic pork products. The muscle fiber is the basic composition unit of the muscle, and the thickness of the muscle fiber is an important factor influencing the eating quality and the processing quality of pork. The muscle fibers can be classified into various types according to different classification methods, and the content of different types of muscle fibers is different between different varieties or different parts of the same variety. In addition, the diameters of different types of muscle fibers are different, for example, the diameter of the fast muscle fiber is larger than that of the slow muscle fiber, which is divided according to the contraction speed of the muscle fiber.
At present, the method for measuring the diameter of muscle fibers mainly comprises the steps of preparing a paraffin section of a skeletal muscle sample, measuring the long axis and the short axis of each muscle fiber randomly through a micrometer or measurement software after HE dyeing, calculating the geometric mean value of the long axis and the short axis or calculating the product of the long axis, the short axis and 0.7, converting the geometric mean value into the diameter of each muscle fiber, and finally obtaining the diameter value of the muscle fiber by measuring a plurality of muscle fibers and then taking the mean value. Under physiological conditions, the cross section of the muscle fiber is close to a circle, but the muscle fiber becomes a polygon or an ellipse after histological treatment, which causes inaccurate measurement of the diameter of the muscle fiber, takes long time for measuring a large number of muscle fibers in sequence, and has low measurement efficiency. In addition, there is also a method in which muscle tissue is soaked in 10% nitric acid, dispersed into individual muscle fibers, the diameter of each muscle fiber is measured under a microscope through an ocular micrometer, and then the average value is taken to obtain the diameter of the muscle fiber. However, the method randomly selects the single muscle fiber to measure sequentially, errors may occur due to different measuring personnel, the type of the muscle fiber which is thin and low in proportion in the muscle may be ignored, and a large number of muscle fibers need to be measured to reduce the errors, so that the time consumption is long, and the measuring efficiency is low. The muscle fiber diameter measurement technology restricts the research of the muscle fiber diameter to a certain extent and limits the progress of the improvement of the pork quality.
Disclosure of Invention
The invention provides a pig muscle fiber diameter measuring method based on Image J Image analysis, which is a method for obtaining a better pig muscle fiber diameter by taking a picture of a prepared paraffin section of skeletal muscle after HE dyeing and processing and analyzing the picture by using Image J, reduces the influence of the change of the muscle fiber form and the muscle fiber type on the muscle fiber diameter result after HE dyeing, avoids the conditions of long time consumption, low measuring efficiency and artificial errors of the existing muscle fiber measurement, can realize quick batch measurement, is simple to operate and solves the problems in the prior art.
The technical scheme adopted by the invention is as follows:
a pig muscle fiber diameter measuring method based on Image J Image analysis comprises the following operation steps:
s1, acquiring images of skeletal muscle fibers of pigs
(1) Collecting a pig skeletal muscle sample;
(2) Preparing skeletal muscle paraffin sections;
(3) HE staining of paraffin sections;
(4) Collecting an image;
s2, carrying out quantitative analysis processing on the Image of the S1 by using Image J software;
s3, calculating the diameter of muscle fiber
The transverse cross-sectional area S1 of the muscle fibers is the total area S0 of the muscle fibers in the selected region divided by the number N of muscle fibers in the selected range, i.e., S1= S0/N;
calculating the average diameter d1, d2, d3 of the muscle fibers in a plurality of selected ranges (n) \8230; dn; the average muscle fiber diameter d = (d 1+ d2+ d3+ \8230; + dn)/n of the subject was obtained.
Further, step S3 is performed to manually select the muscle fiber measurement area and the muscle fiber count in the muscle fiber measurement area, so as to ensure that the total number of the muscle fiber diameters in the plurality of measurement areas is larger than 150.
Further, the Image J software processing in step S2 operates to:
(1) Exporting a high-definition picture which is shot by a microscopic imaging system and is magnified by 200 times to a computer, and opening a myofiber picture to be processed by using Image J software;
(2) Setting Scale parameters by using a software Set Scale tool, and matching pixel points of the picture with actual lengths to obtain the actual micrometer (mum) length of the picture;
(3) Converting the picture type into a gray-scale image with an 8-bit format by using a software type tool;
(4) Changing the Image into a binary Image by using an adjust-Threshold tool under an Image menu, wherein the muscle fibers are black, and the background among the muscle fibers is white;
(5) Filling the meshes in the muscle fibers formed by normal water loss of the muscles in the process of slicing the muscle fibers to black by using a software PaintBrush filling tool;
(6) Selecting a muscle fiber area in the picture by using a Freehand selection tool, and removing incomplete muscle fibers;
(7) Using the Measure tool, the total area S0 (μm) of the muscle fiber was measured under the conditions of Set measures to check the limit to threshold option 2 )；
(8) Counting the number N of muscle fibers in the selected range by using a Cell Counter tool, substituting the number N into a muscle fiber diameter calculation formula, and further calculating to obtain the average muscle fiber diameter of the individual; and calculating the diameters of the muscle fibers of a plurality of regions to obtain the average value of the diameters of the muscle fibers, and ensuring that the total number of the muscle fibers in a plurality of selected ranges is more than 150.
Further, in the step S1, (1) a pig skeletal muscle sample is collected, after a pig is slaughtered, skeletal muscle tissue of a part to be detected is taken, peripheral muscle membrane, tendon, fascia and fat tissue are removed, meat blocks of 1cm × 1cm × 2cm (length × width × height) are taken along the muscle fiber direction, and are rapidly placed in 4% paraformaldehyde fixing solution for fixing;
fixing a skeletal muscle sample for more than 24 hours, cutting the skeletal muscle sample into tissue blocks with the size of 0.5cm multiplied by 0.5cm, putting the tissue blocks into a dehydration box for dehydration, then performing tissue transparency, paraffin dipping, embedding, cooling and wax block trimming, finally cutting the tissue blocks into sections with the thickness of 4 mu m, fishing out after flattening the tissue, baking the sections and storing the sections at normal temperature for later use;
in the step S1, (3) the slices are dewaxed and then subjected to HE dyeing treatment;
and (5) adding a scale to the muscle fiber image during image acquisition in the step (4) of S1, and selecting 200-time magnification for measuring the diameter of the pig fiber.
Further, in step S1, (4) the image capturing operation is performed by an imaging system using microscopy.
Further, in the step S1, in the operation of (4), image acquisition is performed by using Nikon, nikon Eclipse E100 microscopy and Nikon, nikon DS-U3.
Further, the specific operation of (2) preparing the skeletal muscle paraffin section in the step S1 is as follows:
after the skeletal muscle sample is fixed for more than 24 hours, trimming the skeletal muscle sample into tissue blocks with the sizes of 0.5cm multiplied by 0.5cm by using a scalpel, and putting the tissue blocks into a dehydration box; sequentially dehydrating in gradient alcohol by using a DIAPATH dehydrator (Donatello) to ensure that the water in the tissues is completely removed; performing tissue transparency by alcohol benzene for 5-10min, xylene (Chinese medicine, 10023418) I for 5-10min, and xylene II for 5-10 min; putting the slices into 65-degree molten paraffin I1h, 65-degree molten paraffin II 1h and 65-degree molten paraffin III 1h in sequence for tissue waxing; then putting the tissues soaked with the wax into an embedding frame, embedding the tissues in an embedding machine (JB-P5, wuhan Junjie electronic Co., ltd.), cooling the tissues in a-20-degree freezing table (JB-L5, wuhan Junjie electronic Co., ltd.), and finishing a wax block; finally, the cooled wax block is placed in a paraffin slicer (RM 2016) to be sliced into slices with the thickness of 4 microns, the slices float on a spreading machine (KD-P, kinghua, zhejiang province) to flatten tissues at warm water of 40 ℃, after the tissues are fished up by using a glass slide, the slices are baked in a 60 ℃ oven (GFL-230, lai glass instruments, teddy, tianjin), and the slices are taken out to be stored at normal temperature for later use after being baked by water and wax.
Further, the HE dyeing process comprises the following operations:
placing the section into hematoxylin staining solution for staining for 3-5 min to stain cell nucleus, and transferring into water to wash away staining solution and floating color; then placing the slices into hematoxylin differentiation solution (Servicebio, G1039) for differentiation for 2-5 s, removing excessive combined and non-specific adsorbed dyes in tissues, and transferring into running water for full washing; putting the hematoxylin bluing liquid (Servicebio, G1040) for 2-5 s to make the hematoxylin blue, and transferring the hematoxylin bluing liquid into running water for full washing; putting the slices into 85% and 95% alcohol in sequence, dehydrating for 5min respectively, and then putting the slices into eosin dye solution for dyeing for 5-8 min to ensure that the muscle fibers are dyed into deep peach red; placing the slices in anhydrous alcohol I5 min, anhydrous alcohol II 5min, anhydrous alcohol III 5min, dimethyl I5 min, and xylene II 5min in sequence, transparentizing, and sealing with neutral gum
The invention has the beneficial effects that:
1. the prepared paraffin section of skeletal muscle is photographed after HE staining, and the photograph is processed and analyzed by using Image J software, so that the diameters of all muscle fibers contained in a selected area can be simultaneously measured, the total area of 40-70 muscle fibers can be identified at one time, the average value of hundreds of muscle fiber diameters can be obtained by measuring 3-5 visual fields, and the method is a rapid batch measurement method for the diameters of the muscle fibers of skeletal muscle of pigs. The method effectively improves the determination efficiency, avoids artificial measurement errors in measurement of single muscle fibers, and has the advantages of accurate and reliable result, simple operation and easy implementation.
2. The method for analyzing the diameter of the muscle fiber based on the Image J Image utilizes software to count the area of a black area containing a plurality of muscle fibers, utilizes the characteristic that the muscle fibers are close to a round shape in a physiological state, can deduce the diameter of the muscle fiber through a simple round area calculation formula after calculating the average area of the muscle fibers, has simple calculation, and also solves the problem of measurement difficulty and errors caused by irregular change of the dehydrated shape of the muscle fibers in the conventional method for measuring the diameter of the muscle fiber. The method has reliable, accurate and objective results.
3. According to the method, before the Image J is used for processing and analyzing the picture, the specific conditions of the pig skeletal muscle fiber Image acquisition operation are optimized, the muscle fibers are dyed in deep pink color by controlling the dyeing time of the slices in the HE dyeing process, the convenience of subsequent identification and processing by using the Image J is facilitated, the muscle fibers can be converted into black color as far as possible in the binary Image conversion process, errors can be effectively reduced, the influence caused by human factor judgment interference is avoided, and the result is more accurate.
4. When the Image J is used for processing and analyzing the picture, the muscle fiber measuring area is manually selected and the muscle fiber count in the muscle fiber measuring area is ensured to ensure that the total number of the muscle fiber diameters is more than 150 through the measurement in a plurality of areas, all types of muscle fibers contained in the selected area under the physiological state are measured, the influence of the muscle fiber types on the measuring result is reduced, and an objective and accurate muscle fiber diameter result is obtained.
Drawings
FIG. 1 is an original photograph of a paraffin section HE staining of skeletal muscle of a pig;
FIG. 2 is a graph of HE staining 8-bit of a paraffin section of skeletal muscle of a pig according to the present invention;
FIG. 3 is a two-value view of the paraffin section HE staining of porcine skeletal muscle according to the present invention;
FIG. 4 is a schematic representation of a manually selected muscle fiber measurement area of the present invention;
FIG. 5 is a schematic representation of the muscle fiber count within a manually selected muscle fiber measurement area of the present invention;
FIG. 6 is a graph showing the conventional calculation of muscle fiber area by measuring the length of the major and minor axes of muscle fibers;
FIG. 7 is a calculated muscle fiber area when the dehydration voids within the interior of a single muscle fiber are unfilled;
FIG. 8 is a graph of the area of individual muscle fibers measured using the method of the present patent.
Wherein the "+" sign in figure 5 is shown as the counted muscle fibers.
Detailed Description
In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.
The method selects the longissimus dorsi tissue of the Du-Long three-dimensional pig, and samples are taken to determine the diameter of skeletal muscle fiber of the pig. The Du-Chang three-element pig feeds complete feed according to the nutritional requirements of different days of age, provides free drinking water, and is slaughtered when the weight reaches 110 kg. The specific experimental process is as follows:
1. porcine skeletal muscle sample collection
After the pig is slaughtered, taking skeletal muscle tissues of a part to be detected, removing peripheral sarcolemma, tendon, fascia and adipose tissues, taking meat blocks of 1cm multiplied by 2cm (length multiplied by width multiplied by height) along the muscle fiber direction, and quickly placing the meat blocks in 4% paraformaldehyde fixing solution for fixing for later use;
2. preparation of skeletal muscle Paraffin section
Fixing the pig skeletal muscle sample for 28h, cutting into tissue blocks with the size of 0.5cm multiplied by 0.5cm by using a scalpel, and placing the tissue blocks into a dehydration box; the dehydration was carried out in gradient alcohol using a DIAPATH dehydrator (Donatello) in sequence, specifically: 4h of 75% alcohol, 2h of 85% alcohol, 2h of 90% alcohol, 1h of 95% alcohol, 30min of absolute ethyl alcohol (Chinese medicine, 100092683) I and 30min of absolute ethyl alcohol II, so as to ensure that the water in the tissue is completely removed; making the tissue transparent by alcohol benzene 5-10min, xylene (Chinese medicine, 10023418) I5-10 min, and xylene II 5-10 min; putting the slices into 65-degree molten paraffin I1h, 65-degree molten paraffin II 1h and 65-degree molten paraffin III 1h in sequence to carry out tissue waxing; then, the tissues soaked with the wax are placed in an embedding frame, embedded in an embedding machine (JB-P5, junjie electronic Co., ltd., wuhan), cooled in a-20-degree freezing table (JB-L5, junjie electronic Co., ltd., wuhan), and the wax block is trimmed; finally, the cooled wax block is placed in a paraffin slicer (RM 2016) to be sliced into 4 μm thick slices, the slices float on a spreading machine (KD-P, kinghua Kedi, zhejiang province) to flatten the tissue at 40 ℃ warm water, after the tissue is fished out by using a glass slide, the slices are baked in a 60 ℃ oven (GFL-230, lai glass instrument, tengjin) to dry the wax, and the wax is baked and taken out to be stored at normal temperature for later use.
3. HE staining of Paraffin sections
Placing the prepared pig skeletal muscle paraffin section into dimethylbenzene I20 min, dimethylbenzene II 20min, absolute ethyl alcohol I5 min, absolute ethyl alcohol II 5min and 75% alcohol 5min in sequence, washing with water, and dewaxing the section.
Section staining was performed using HE dye set (Servicebio, G1003). Placing the section into hematoxylin staining solution for staining for 3-5 min to stain cell nucleus, and transferring into water to wash away staining solution and floating color; then placing the slices into hematoxylin differentiation solution (Servicebio, G1039) for differentiation for 2-5 s, removing excessive combined and non-specific adsorbed dyes in tissues, and transferring into running water for full washing; putting the hematoxylin bluing liquid (Servicebio, G1040) for 2-5 s to make the hematoxylin blue, and transferring the hematoxylin bluing liquid into running water for full washing; dewatering the slices by 85% and 95% alcohol in sequence for 5min respectively, and then dyeing in eosin dye solution for 5-8 min to ensure that the muscle fiber is dyed to be deep pink, so that Image J is conveniently used for Image recognition and conversion; placing the slices in anhydrous alcohol I5 min, anhydrous alcohol II 5min, anhydrous alcohol III 5min, dimethyl I5 min, and xylene II 5min in sequence, making transparent, and sealing with neutral gum (Chinese medicine, 10004160).
4. Image acquisition
Image acquisition was performed by an imaging system (Nikon, nikon DS-U3) using a microscope (Nikon, nikon Eclipse E100) microscopy, and a ruler was added to the muscle fiber image. The diameter of the porcine muscle fibers is measured with the optimal magnification of 200 times, so that the integrity of the muscle fibers in the selected area can be ensured when the range is manually drawn by using Image J software.
5. Image processing procedure using Image J software
1) Exporting a high-definition picture which is shot by using a microscopic imaging system and is magnified by 200 times to a computer, and opening a myofiber photo to be processed by using Image J software, wherein the myofiber photo is shown in figure 1;
2) Setting Scale parameters by using a Set Scale tool under a software Analyze menu: distance in pixels is 234 knock Distance is 100.00, pixel aspect is 1.0, unit of length is mum, global option is selected, pixel points of the picture are matched with actual length, and the actual micrometer (mum) length of the picture is obtained;
3) Converting the picture type into a gray-scale Image with an 8-bit format by using a type tool under a software Image menu, as shown in FIG. 2;
4) Adjusting an Image Threshold value by using an adjust-Threshold tool under an Image menu, enabling muscle fibers to be red by adjusting a scroll bar under a Threshold window, ensuring that the background among the muscle fibers is kept white, clicking application, and changing the Image into a binary Image after application, wherein the muscle fibers in the Image are black, and the muscle fibers are white, as shown in fig. 3;
5) Filling the meshes formed by muscle dehydration in the process of making the muscle fiber into black by using a PaintBrush filling tool, and adjusting the Brush width to be 40 and other larger widths in the Brush Options for convenient and quick filling;
6) Selecting a muscle fiber area in the picture by using a Freehand selection tool, removing incomplete muscle fibers and ensuring that the muscle fibers in the selected area can be observed into a complete cross section as shown in figure 4;
7) The limit to threshold is selected in the Set measures option under the Analyze menu, and then the total area S0 of the black muscle fiber in the selected area is automatically measured to be 197128.35 (mum) by using the Measure tool 2 )；
8) The number of muscle fibers N1=69 within the selected range was counted using the Cell Counter tool, as shown in fig. 5.
6. Calculation of muscle fiber diameter
The transverse area A of the muscle fibers is the total area A0 of the muscle fibers in the selected area divided by the number N of muscle fibers in the selected area, i.e., S1 (μm) 2 )＝S0/N＝197128.351/69＝2856.93。
The mean diameter of the muscle fibers in a number of fields of view (N = 3) d1=60.33 μm (N1 = 69), d2=63.22 μm (N2 = 57), d3=59.65 μm (N3 = 71) was calculated, and the mean muscle fiber diameter of the individual d = (d 1+ d2+ d3+ \ 8230; \8230;/N = (60.33 +63.22+ 59.65)/3 =61.67 μm.
When the Image J is used for analyzing the muscle fiber picture dyed by the HE, the invention overcomes the errors caused by firstly measuring the long axis and the short axis of each muscle fiber and then calculating the area of the muscle fiber by using the method of the long axis multiplied by the short axis multiplied by 0.7 and deducing the diameter of the muscle fiber when the traditional method is used for measuring, and as shown in figure 6, the calculated muscle fiber area is 68.519 multiplied by 55.271 multiplied by 0.7=2650.979554; the total area of the muscle fiber which is converted into black can be quickly identified by converting the picture into an 8-bit picture and then into a binary picture through software. During measurement, in order to reduce the influence of the gap after the muscle fiber loses water on the measurement in the paraffin section manufacturing process (fig. 7), a filling tool of software is utilized to fill the gap after the muscle fiber loses water to black, and the gap after the muscle fiber loses water is added into the area of the muscle fiber, so that the cross-sectional area of the muscle fiber in a real physiological state is maximally restored, as shown in fig. 8.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.
Claims (6)
1. A pig muscle fiber diameter measuring method based on Image J Image analysis is characterized by comprising the following operation steps:
s1, acquiring images of skeletal muscle fibers of pigs
(1) Collecting a pig skeletal muscle sample;
(2) Preparing skeletal muscle paraffin sections;
(3) HE staining of paraffin sections;
(4) Collecting an image;
s2, carrying out quantitative analysis processing on the Image of the S1 by using Image J software;
s3, calculating the diameter of muscle fiber
The transverse cross-sectional area S1 of the muscle fibers is the total area S0 of the muscle fibers in the selected region divided by the number N of muscle fibers in the selected range, i.e., S1= S0/N;
diameter of muscle fiber
Calculating the average diameter d1, d2, d3 of the muscle fibers in a plurality of selected regions (n) \8230; dn; the average muscle fiber diameter d = (d 1+ d2+ d3+ \8230; + dn)/n of the subject was obtained.
2. The method for measuring the diameter of the pig muscle fibers based on Image J Image analysis as claimed in claim 1, wherein step S3 is performed to manually select the muscle fiber measuring area and the muscle fiber count in the muscle fiber measuring area, so as to ensure that the total number of the muscle fiber diameters is more than 150 after a plurality of areas are measured.
3. The method for measuring the diameter of the pig muscle fiber based on Image J Image analysis as claimed in claim 1, wherein the Image J software processing in the step S2 is:
(1) Exporting a high-definition picture which is shot by a microscopic imaging system and is magnified by 200 times to a computer, and opening a myofiber picture to be processed by using Image J software;
(2) Setting Scale parameters by using a software Set Scale tool, and matching pixel points of the picture with actual lengths to obtain the actual micrometer (mum) length of the picture;
(3) Converting the picture type into a gray-scale image with an 8-bit format by using a software type tool;
(4) Adjusting the image Threshold value by using a software adjust-Threshold tool, converting the image into a binary image after application, wherein the muscle fibers are black, and the background among the muscle fibers is white;
(5) Filling meshes in the muscle fibers formed by muscle dehydration in the process of slicing the muscle fibers to black by using a software filling tool;
(6) Selecting a muscle fiber area in the picture by using a manual selection tool, and removing incomplete muscle fibers;
(7) Measuring the area of black region in the manually selected region by using a measuring tool to obtain the total area S0 (mum) of muscle fiber 2 )；
(8) And (3) counting the number N of the muscle fibers in the selected range by using a Cell Counter tool, substituting the number N into a muscle fiber diameter calculation formula, and further calculating to obtain the average muscle fiber diameter of the individual, wherein the total number of the muscle fibers in the plurality of selected ranges is more than 150.
4. The method for measuring the diameter of the pig muscle fiber based on Image J Image analysis according to claim 1, wherein in the step S1, (1) the sample of the pig skeletal muscle is collected, after the pig is slaughtered, the skeletal muscle tissue of the part to be detected is taken, the peripheral sarcolemma, tendon, fascia and fat tissue are removed, and then 1cm x 2cm (length x width x height) meat blocks are taken along the muscle fiber direction and are rapidly placed in 4% paraformaldehyde fixing solution for fixation;
fixing a skeletal muscle sample for more than 24 hours, cutting the skeletal muscle sample into tissue blocks with the size of 0.5cm multiplied by 0.5cm, putting the tissue blocks into a dehydration box for dehydration, then performing tissue transparency, paraffin dipping, embedding, cooling and wax block trimming, finally cutting the tissue blocks into sections with the thickness of 4 mu m, fishing out after flattening the tissue, baking the sections and storing the sections at normal temperature for later use;
in the step S1, (3) the slices are dewaxed and then subjected to HE dyeing treatment;
and (5) adding a scale to the muscle fiber image during image acquisition in the step (4) of S1, and selecting 200-time magnification for measuring the diameter of the pig fiber.
5. The method for measuring the diameter of the pig muscle fiber based on Image J Image analysis according to claim 4, wherein the Image acquisition in step S1 (4) is performed by an imaging system by microscopic examination.
6. The method for determining the diameter of the pig muscle fiber based on Image J Image analysis as claimed in claim 4, wherein the HE staining process comprises the following operations:
placing the section into hematoxylin staining solution for staining for 3-5 min to stain cell nucleus, and transferring into water to wash away staining solution and floating color; then putting the slices into hematoxylin differentiation solution for differentiation for 2-5 s, removing excessive bound and non-specifically adsorbed dyes in tissues, and transferring the tissues into running water for full washing; adding hematoxylin bluing liquid for 2-5 s to make hematoxylin blue, and washing in flowing water; the slices are put into 85 percent and 95 percent alcohol in sequence for dehydration for 5min respectively, and then are put into eosin dye liquor for dyeing for 5-8 min, so as to ensure that the muscle fibers are dyed into deep peach-red; placing the slices in anhydrous alcohol I for 5min, anhydrous alcohol II for 5min, anhydrous alcohol III for 5min, dimethyl I for 5min, and xylene II for 5min, making transparent, and sealing with neutral gum.
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