CN113075408A - Immunohistochemical quantitative method using histone as internal reference - Google Patents
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Abstract
The invention provides an immunohistochemical quantitative method using histone as an internal reference. The method adopts histone family as internal reference protein of immunohistochemistry, and utilizes immunohistochemistry method and image processing software to carry out quantitative analysis on the protein to be detected; in addition, the method may further comprise the step of separately staining H3F3B and the tumor marker or the step of simultaneously staining H3F3B and the tumor marker using a two-color immunohistochemical method. The immunohistochemical quantitative method using histone as internal reference provided by the invention utilizes the uniformity of protein level of histone in longitudinal and transverse sections of tissue and different parts of the same section, can overcome the difference of factors such as sample preparation, antigen incubation condition, primary antibody and secondary antibody incubation condition, has stable and highly repeatable result, and can greatly improve the quantitative accuracy of IHC marker in life science and immunopathology.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an immunohistochemical quantitative method using histone as an internal reference.
Background
Proteins are involved in many biological processes and in the ultimate executives of all biological functions. Many proteins whose expression changes in disease can serve as drug targets or biomarkers. Therefore, accurate measurement and detection of protein expression is critical to adequately describe these functions. Therefore, detection and quantification of proteins are extremely important. Conventional techniques, such as ELISA, immunoblotting (WB), targeted mass spectrometry, etc., have facilitated protein detection and quantification studies in biological samples. Furthermore, Immunohistochemistry (IHC) is also an important method for detecting proteins and their cellular localization and expression levels in tissues and cells.
Among them, immunohistochemistry is commonly used in surgery and clinical pathology because it is crucial for diagnosis and prediction of prognosis and treatment response. IHC is also a simple and effective method to determine the expression levels and cellular localization of tumor markers and has recently also been used to detect prognostic and predictive biomarkers (e.g. Her2 and PD-L1) for personalized cancer treatment. However, many parameters in the sample preparation, IHC experiments, post-analytical stages all affect the final result. Furthermore, the results of IHC are usually assessed non-quantitatively by a pathologist and scored according to staining intensity and the cellular rate of positive expression and correct intracellular localization, so that the results are only relative and not absolute.
Generally, Her2 and PD-L1 scores evaluated by pathologists help to select treatment for patients, and many suggestions or guidelines on how to score Her2 or PD-L1 IHC are disclosed, but the results scored by pathologists vary, even by more than 25%. Although the extensive use of fully automated immunohistochemical instruments, full-sided imaging systems, digital image analysis software, greatly improves the quantification of IHC and researchers have now made much effort to quantify IHC results (e.g., northern european immunohistochemical quality control), the lack of standardization and reference controls that make reproducibility poor remains a major problem.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an immunohistochemical quantitative method using histone as an internal reference. The members of the histone family are mostly localized in the nucleus, and are easy for IHC quantification; it is stably and uniformly expressed in 20 types of human cancer tissues of protein type, and uniformly expressed in the longitudinal and transverse directions and inside of tumor tissues, showing its potential role as an internal reference.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an immunohistochemical quantification method using histone as an internal reference; the method adopts histone family as internal reference protein of immunohistochemistry, and utilizes immunohistochemistry method and image processing software to carry out quantitative analysis on the protein to be detected.
Furthermore, after the H-Score is obtained by using image processing software, the ratio of the protein H-Score to be detected to the histone H-Score is calculated, and then the protein to be detected is subjected to quantitative analysis.
Further, the image processing software includes a HALO digital pathology image software.
Further, the histone is selected from one of H1F0, H2B and H3F 3B.
Further, the histone is H3F 3B.
Further, the method comprises the step of staining H3F3B and the protein to be tested, respectively.
Further, the method comprises the step of simultaneously staining H3F3B and the protein to be detected by a two-color immunohistochemical method.
Further, the method adopts the dilution ratio of the H3F3B primary antibody of 1: (200-800).
Further, the method adopts the dilution ratio of the H3F3B primary antibody of 1: 400.
in a second aspect, the present invention provides the use of histone proteins as an internal reference in a method for the quantitative analysis of tumor markers using immunohistochemistry.
Further, the above tumor markers include PGM1, Her2 and PD-L1.
Further, the method comprises a step of staining the H3F3B and the tumor marker separately or a step of staining the H3F3B and the tumor marker simultaneously by a two-color immunohistochemical method.
In a third aspect, the present invention provides a software for pathological image analysis based on the above method, the software includes a software program for obtaining the ratio of the protein H-Score to be tested to the histone H-Score.
In a fourth aspect, the present invention provides a kit for quantitative analysis of a protein to be tested using histone as an internal reference, the kit comprising reagents and software for use in the operation of the above method. By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the immunohistochemical quantitative method using histone as internal reference provided by the invention utilizes the uniformity of protein level of histone in longitudinal and transverse sections of tissue and different parts of the same section, can overcome the difference of factors such as sample preparation, antigen incubation condition, primary antibody and secondary antibody incubation condition, has stable and highly repeatable result, and can greatly improve the quantitative accuracy of IHC marker in life science and immunopathology.
Drawings
Figure 1 shows the feasibility of the histone family as IHC reference proteins; wherein, panel a shows immunohistochemical images of 20 human cancer tissues; FIGS. b-d show immunohistochemical scores for H1F0, H2B, and H3F3B, respectively, in each cancer tissue section;
figure 2 shows the specificity of selected H3F3B antibodies; wherein panel a shows the results of a western blot using a sample of hepatocellular carcinoma (HCC) to verify the specificity of selected H3F3B antibodies; FIGS. b-d show the results of antibody uptake experiments using 0. mu.g, 5. mu.g and 15. mu. g H3F3B antigen, respectively;
FIG. 3 shows the results of the quantification of H3F3B using the immunohistochemical method; wherein, a is an original picture of staining H3F3B after immunohistochemistry, b is an image calculated by HALO software, and c is a statistical result graph of the number of negative cells (0+ cells) and the number of positive cells (1+ cells, 2+ cells, 3+ cells) obtained by adopting the HALO software;
FIG. 4 shows the results of staining hepatoma cells and paracancerous liver tissues and immunohistochemical scoring using antibodies at different dilution ratios; wherein panel a shows representative images of HCC tissue staining with H3F3B antibody at different dilution ratios; FIG. b is a graph showing H-score results of HCC tissues of 45 patients with liver cancer; FIG. c shows the average H-score of different H3F3B antibody dilutions in HCC tissues from 45 patients with liver cancer; panel d shows representative images of staining of paracancerous tissue (Adj) with H3F3B antibody at different dilution ratios; FIG. e is a graph representing the results of H-score in tissues adjacent to 45 patients with liver cancer; FIG. F shows the average values of H-score at different dilution ratios of H3F3B antibody in the paracarcinoma tissues of 45 patients with liver cancer;
fig. 5 shows by IHC staining results that spatial heterogeneity does not affect H3F3B protein expression; wherein, panel a shows the results of immunohistochemical scoring of wax masses for 9 hepatocellular carcinoma patients, each of which was cut into 4 pieces and stained with H3F3B antibody; panel b shows immunohistochemical scoring results of H3F3B staining of five different regions of each tumor section; panel c shows immunohistochemical scoring results of H3F3B staining of five tumor regions of the same section;
FIG. 6 shows the results of the use of H3F3B as an internal reference in the analysis of other markers; wherein, the graph a shows the result of staining PGM1 and H3F3B in different sections; panel b shows the H-Score ratio of PGM1 and H3F3B in panel a; FIG. c shows the results of staining for Her2 and H3F3B in different sections, respectively; FIG. d shows the correspondence of the Her2/H3F3B ratio with the pathologist's scoring results; panel e shows the distribution of pathologist scoring results versus Her2/H3F3B ratio; FIG. F shows the results of staining PD-L1 and H3F3B in different sections, respectively; panel g shows the ratio of H-Score (PD-L1H-Score/H3F3B H-Score) to PD-L1 positive cells (R)20.9948) is highly correlated; panel H shows that the H-Score ratio (PD-L1H-Score/H3F3B H-Score) is highly consistent with the pathologist's Score;
fig. 7 shows the results of IHC two-color staining with Her2 and H3F3B for quantification of Her2 indoors and from room to room; wherein, fig. a shows a picture of IHC two-color staining with Her2 and H3F 3B; FIG. b shows a graph comparing the results of H-Score ratio (Her 2H-Score/H3F 3B H-Score) for day 1, day 3 and external laboratory tests; FIG. c shows the correspondence of the Her2/H3F3B ratio with the pathologist's scoring results; FIG. d is a comparison of the Her2/H3F3B ratios calculated after scanning with the first slice on a KFBio KF-PRO-005 scanner and a Hamamatsu Nano S60 scanner, respectively;
FIG. 8 shows the Her2/H3F3B ratio of DC-IHC and the high uniformity of staining alone.
Detailed Description
The invention provides a novel method for quantifying the protein expression level by IHC by taking histone H3F3B as an internal reference standard, which has high accuracy and repeatability. Using H3F3B as an internal reference, Her2 and PD-L1 expression levels were accurately assessed with high consistency by current methods (scores assessed by pathologists). Furthermore, the inventors also demonstrated the feasibility of H3F3B as a quality control inside and between laboratories.
Specifically, the immunohistochemical quantification method using histone as an internal reference; the method adopts histone family as internal reference protein of immunohistochemistry, and utilizes immunohistochemistry method and image processing software to carry out quantitative analysis on the protein to be detected.
In a preferred embodiment of the present invention, after the H-Score is obtained by using the image processing software, the ratio of the H-Score to the histone H-Score of the protein to be detected is calculated, and the protein to be detected is quantitatively analyzed.
In a preferred embodiment of the present invention, the image processing software includes, but is not limited to, a HALO digital pathology image software.
In a preferred embodiment of the invention, the histone is one selected from the group consisting of H1F0, H2B and H3F 3B.
In a preferred embodiment of the present invention, the histone is H3F 3B.
In a preferred embodiment of the present invention, the method comprises the step of staining H3F3B and the protein to be tested, respectively.
In a preferred embodiment of the present invention, the method comprises the step of staining H3F3B and the protein to be tested simultaneously by a two-color immunohistochemical method.
In a preferred embodiment of the present invention, the dilution ratio of the H3F3B primary antibody used in the above method is 1: (200-800).
In a preferred embodiment of the present invention, the dilution ratio of the H3F3B primary antibody used in the above method is 1: 400.
the present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Materials and reagents
1. Patient and tumor tissue
HCC, adjacent liver tissue samples, and breast cancer tissue were collected from patients with liver cancer or breast cancer and approved by the institutional review Board of the Dong Gangdan surgical Hospital (EHBH; approval No.: EHBHKY 2014-03-006). All patients received written informed consent.
2. Reagent
Reagents used in Western immunoblotting (Western Blot): H3F3B antibody (concentration 1: 1000, rabbit polyclonal antibody, GeneTex, GTX115549), secondary antibody: goat anti-mouse IgG (H + L) -HRP conjugate (concentration 1: 5000; using # 170-.
Reagents used in two-color immunohistochemistry (DC-IHC) experiments: H3F3B antibody (concentration 1: 50, mouse monoclonal antibody, positive organisms, 250011), Her2 antibody (concentration 1: 50; rabbit monoclonal antibody, Shanghai Long island antibody diagnostics, Inc., M-0196); the secondary antibody adopts alkaline phosphatase coupling goat anti-rabbit IgG (concentration 1: 100, Drosmidd biotechnology, BA1011) and goat anti-mouse IgG (H + L) -HRP conjugate (concentration 1: 100, Bio-Rad, 170-; DAB color kit (GK500710, Gene technology) was used for H3F3B staining, Fast-Red (ZLI-9045, ZSBBio) was used for Her2 staining.
General procedure
1. Slice preparation and immunohistochemistry
Two pathologists with abundant experience accurately diagnose the HE staining result and then mark lesions in corresponding wax blocks in advance; taking a cylinder with the diameter of 1.5mm from the marked tissue by using a tissue puncture needle, and preparing a tissue chip after putting a pre-prepared wax block; the tissue chip or the conventional slice has a thickness of 4 μm, is placed on the slice coated with 3-aminopropyltriethoxysilane, and is baked at 90 deg.C for 1 hour for IHC; dewaxing in xylene for 2 times each for 10min, and then hydrating with a gradient ethanol solution (100%, 95%, and 85% each for 5 min); after boiling in EDTA buffer at pH 9.0, antigen retrieval was performed by keeping at 100 ℃ for 10min (H3F3B, Her2, PGM1 and DC-IHC) or 20 min (PD-L1) and cooling at room temperature for 60 min; using 3% H2O2Incubate for 30min to block endogenous peroxidase activity, and finally block non-specific binding sites with 5% BSA/PBS.
2. Scanning and calculating immunohistochemical staining results
After immunohistochemical staining was complete, images were saved using a scanner, including KFBio KF-PRO-005(403WSIS,. times.40, 0.238 μm/pixel) and Hamamatsu Nano zoom S60(1832WSIs,. times.40, 0.220 μm/pixel). All slices were scanned using a KFBIO KF-PRO-005-EX digital scanner. Quantitative analysis for H-Score was performed using the Multiplex IHC v2.3.4HALO algorithm (Indica Labs) of HALO software, with the following main settings: DAB nuclear (optical density) positivity threshold of H3F3B of 0: 0-0.099; 1+: 0.1-0.299; 2+ 0.3-0.499; 3+ 0.5-2.5; the optical densities of PGM1, Her2 and PD-L1 are: 0:0 to 0.099; 1+: 0.1-0.299; 2+: 0.3-0.499; 3+: 0.5-2.5. The H-Score was calculated as follows: H-Score ═ 1 × 1+ cells) + (2 × 2+ cells) + (3 × 3+ cells).
Example 1
This example demonstrates the feasibility of the histone family as IHC reference protein, the specific procedures and results are as follows:
1. IHC image data were downloaded from the Human Protein Atlas (https:// www.proteinatlas.org /) website and analyzed by Halo software (Indica Lab) for H1F0, H2B, H3F3B Protein expression levels (2 tumor sites per patient) in 20 Human cancer (breast, carcinoid, cervical, colorectal, endometrial, glioma, head and neck, liver, lung, lymphoma, melanoma, ovarian, pancreatic, prostate, renal, skin, gastric, testicular, thyroid, and urothelial) tissues, as shown in FIGS. 1 a-d. As can be seen from fig. 1, H1F0, H2B, and H3F3B are stably and uniformly expressed in 20 human cancer tissues, and it was preliminarily confirmed that H1F0, H2B, and H3F3B can serve as internal references of IHC.
2. To further confirm the feasibility of the histone family as an IHC internal reference, the inventors validated the specificity of the selected H3F3B antibody using Western Blot and antibody uptake assays:
2.1Western Blot: firstly, taking 50mg liver cancer tissues to extract protein samples, and then, adopting a BCA protein quantitative reagent (Biyuntian) to carry out protein quantification; secondly, the extracted protein sample is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by adopting a conventional method, and then is transferred to a PVDF membrane (Millipore); dissolving skimmed milk powder in TBS-T buffer (0.05% (v/v) Tween 20/Tris-buffer) with pH of 7.4 to obtain fat-free milk solution, wherein the concentration of the skimmed milk powder is 5% (w/v), adding the fat-free milk solution into the protein sample, adding primary antibody after 1h, incubating overnight at 4 ℃, washing the membrane for 3 times with TBS-T, adding horseradish peroxidase (HRP) -labeled secondary antibody, and incubating for 1h at normal temperature; and fourthly, the protein is developed by a conventional method and analyzed, and the result is shown in figure 2a, and the H3F3B antibody recognizes the 17kDa (position corresponding to H3F 3B) protein.
2.2 antibody uptake assay: after reacting with gradient H3F3B antigen amount (0 μ g, 5 μ g, 15 μ g, GTX115549-pro) and 0.5 μ g H3F3B antibody (reaction system volume is 200 μ l, GTX115549) at 4 ℃ overnight, respectively, 3 serial sections were incubated with H3F3B antibody as primary antibody, respectively, since the antibody adsorbed by the antigen can not react with the antigen in the tissue any more when the H3F3B antigen is excessive, the result should be negative or the positive signal should be reduced when the antibody adsorbed by the antigen is used as an immunohistochemical primary antibody; specific results are shown in FIGS. 2 b-d. As shown in FIGS. 2b-d, immunohistochemical staining was strong when the amount of H3F3B antigen was 0. mu.g; when the antigen amount of H3F3B is 5 mug, immunohistochemical staining is obviously weakened; when the amount of H3F3B antigen was 15. mu.g, almost no staining was observed.
3. H3F3B was stained by immunohistochemistry followed by H-score, the results of which are shown in FIG. 3.
Example 2
This example explores the optimal dilution ratio of the H3F3B antibody as IHC reference protein, and the specific procedures and results are as follows:
H3F3B antibody was diluted 1/200, 1/400 and 1/800 fold (GTX115549), immunohistochemical staining of 45-fold hepatocellular carcinoma and paracancerous liver tissue was performed until no further staining followed by mounting, and scanning was performed on a KFBio KF-PRO-005(403WSIs,. times.40, 0.238 μm/pixel) scanner; H-Score was performed using HALO Multiplex IHC v2.3.4HALO algorithm (indica labs) and plotted as shown in FIGS. 4a and 4 b.
As can be seen in FIGS. 4a-b, the appropriate dilution ratio of H3F3B antibody used in IHC is 1/400.
Example 3
In this example, whether the expression of H3F3B protein is affected by spatial heterogeneity is investigated, and the specific experimental methods and procedures are as follows:
(1) paraffin-embedded tissues of 9 patients were taken, after cutting the first piece of tissue, after further longitudinally cutting 3 pieces of tissue at 20um intervals, H3F3B staining was performed until no further deep staining was performed, scanning was performed, and H-Score was calculated in HALO software, with the results shown in FIG. 5 a;
(2) taking paraffin-embedded tissues of 9 patients, optionally taking 5 tissues (A, B, C, D and E) in each case, cutting each tissue, staining the sections obtained from 5 different areas with H3F3B until no deep staining is performed, scanning, and calculating H-Score in HALO software, wherein the result is shown in FIG. 5B;
(3) 5 different areas were taken from the same section of the section, and after staining H3F3B was performed on the 5 different areas of the image until no more deep staining was observed, the image was scanned and H-Score was calculated by HALO software, and the results are shown in FIG. 5 c.
The results clearly show that the Coefficient of Variation (CV) values for H3F3B in 4 sections were very small with a maximum CV of 0.22% (fig. 5a), and thus the expression of H3F3B was not affected by tumor longitudinal spatial heterogeneity; furthermore, the inventors selected H3F3B staining of five different regions of each tumor section for calculation and found that the CV value was still very small, with a maximum CV of 0.21% (fig. 5 b); the expression of H3F3B was also very consistent in different tumor regions of the same section (fig. 5c), indicating the possibility of H3F3B protein as an IHC internal reference.
Example 4
To date, the expression level assessment of IHC tumor markers is still only semi-quantitative and poorly reproducible due to the lack of quantifiable IHC internal controls. To further evaluate the possibility of H3F3B as an internal reference for IHC quantification, this example utilized PGM1 (representative of nuclear and membrane expression), Her2 and PD-L1 (representative of membrane expression) as IHC markers for prognosis and guidance of treatment, specific protocols and results are as follows:
(1) PGM1 and H3F3B were immunohistochemically bound until no more deep staining (staining results are shown in FIG. 6 a), scanned, H-Score calculated, PGM1/H3F3B ratio calculated, X-Tile software calculated optimal threshold, SPSS software survival analysis, and results are shown in FIG. 6 b.
The results showed that in 285 cases, 204 cases had a low recurrence rate, and 81 cases had a high recurrence rate, and the H-Score ratio of 0.16 was critical for determining the probability of recurrence.
(2) After immunohistochemistry for Her2 and H3F3B until no more deep staining (staining results are shown in FIG. 6), scanning was performed, H-Score was calculated, the ratio of Her2/H3F3B was obtained, plotting was performed by GraphPad software, and the consistency with the pathologist's scoring results was compared (FIG. 6 d). Wherein, as shown in FIG. 6e, the H-Score ratios of Her2 and H3F3B fall into four categories: (1)0 and 1+: the ratio is more than or equal to 0.028; (2)2+: 0.13< the ratio is less than or equal to 0.36; and (3) 3+: the ratio is > 0.44.
As a result, the ratio of H-Score was found to be extremely consistent with the Score of a pathologist.
(3) After immunohistochemistry was performed on PD-L1 and H3F3B respectively until no deep staining was performed, scanning was performed, H-Score was calculated (FIG. 6F), correlation analysis was performed with the positive proportion of PD-L1 after the ratio of PD-L1/H3F3B was obtained (FIG. 6g), and consistency observation with the Score result of a pathologist was performed, and the result is shown in FIG. 6H.
The results showed that the ratio of H-Score (PD-L1H-Score/H3F3B H-Score) and PD-L1 positive cells (R)20.9948) and highly consistent with the pathologist's score.
Example 5
In this example, Her2 was quantified by a two-color immunohistochemical method (DC-IHC), and the specific procedures and results were as follows:
the mixed solution of the H3F3B antibody and the Her2 antibody is a primary antibody, and the secondary antibody mixed solution adopts alkaline phosphatase coupled goat anti-rabbit IgG and goat anti-mouse IgG (H + L) -HRP conjugate; after the primary antibody and the secondary antibody are applied, firstly, Fast-Red staining Her is carried out for 215 minutes, then, washing is carried out for 2 times, then, DAB reagent in a DAB detection kit is used for staining H3F3B until no deep staining is carried out, and then, the piece is sealed; the staining results are shown in FIG. 7 a.
By utilizing the method, 3 continuous sections are adopted, wherein the first section is dyed for the first day, the second section is dyed for the third day, and the third section is dyed for an external laboratory and is sealed; scanning, calculating H-Score, and then obtaining a ratio of Her2/H3F3B, wherein the result is shown in FIG. 7 b; and consistent observation with the scoring result of the pathologist is made, and the result is shown in fig. 7 c; in addition, the first slice was compared to the Her2/H3F3B ratio after scanning with a KFBio KF-PRO-005(403WSIs,. times.40, 0.238 μm/pixel) scanner and a Hamamatsu Nano S60(1832WSIs,. times.40, 0.220 μm/pixel) scanner, respectively (FIG. 7 d).
Comparison of the staining results at different times and comparison of the staining results between chambers (FIG. 7b) shows that the ratio Her2/H3F3B is constant; the distribution of pathologist scored results and double-stained Her2/H3F3B ratios (fig. 7c) showed that the H-Score ratios for Her2 and H3F3B were classified into three categories: the ratio of Her2/H3F3B is divided into three categories: (1)0 and 1+: 0< the ratio is less than or equal to 0.1; (2)2+: 0.19 is less than or equal to 0.44; and (3) 3+: a ratio > 0.46; the results of the scans by the different scanners (FIG. 7d) show that the ratio Her2/H3F3B is relatively constant.
In addition, the inventors compared Her2/H3F3B ratios for the two-color stain and the separate stain of example 4, and the results are shown in FIG. 8. As a result, it was found that the Her2/H3F3B ratio (day 1 data in FIG. 7b) from DC-IHC and the individual staining (data in FIG. 6d) were also highly consistent.
In summary, the IHC two-color staining method using Her2 and H3F3B strictly considers the H3F3B protein as a true internal reference, so this method is an excellent indoor and interventricular quality control method for Her2 quantification.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (11)
1. An immunohistochemical quantitative method using histone as internal reference is characterized in that a histone family is used as the internal reference protein of immunohistochemistry, and the protein to be detected is subjected to quantitative analysis by using immunohistochemical method and image processing software.
2. The method of claim 1, wherein the protein to be detected is quantitatively analyzed by calculating the ratio of the H-Score to the histone H-Score after the H-Score is obtained by using image processing software.
3. The method of claim 1, wherein the histone protein is selected from one of H1F0, H2B, and H3F 3B; preferably H3F 3B.
4. The method of claim 3, comprising the step of separately staining H3F3B and the protein to be tested.
5. The method of claim 3, comprising the step of staining H3F3B and the protein to be tested simultaneously using a two-color immunohistochemical method.
6. The method according to claim 4 or 5, wherein the dilution ratio of the H3F3B primary antibody is 1: (200- > 800); preferably 1: 400.
7. the use of histone as an internal reference in a method for the quantitative analysis of tumor markers using immunohistochemistry.
8. The use according to claim 7, wherein the tumor markers comprise PGM1, Her2 and PD-L1.
9. The use according to claim 7, wherein said method comprises a step of staining H3F3B and the tumor marker separately or a step of staining H3F3B and the tumor marker simultaneously using a two-color immunohistochemical method.
10. Software for pathological image analysis based on the method according to any one of claims 1 to 6, comprising a software program for obtaining the ratio of the protein H-Score to the histone H-Score to be tested.
11. A kit for quantitative analysis of a protein to be tested using histone as an internal reference, said kit comprising reagents and software for use in the process according to any one of claims 1 to 6.
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