IHC

1. Target

This method is a guide to use the Elabscience® RTU antibody for IHC correctly with BOND™MAX (Leica) for immunohistochemical operation and obtain the optimal results.

2. Limits

This method is suitable for the automatic immunohistochemical staining experiment on BOND™ MAX (Leica) instrument with the Elabscience® RTU antibody for IHC.

3. Experimental Steps

1) Reagents and Consumables

1.1 Transparent dewaxing solution.

1.2 Absolute ethanol.

1.3 95% ethanol.

1.4 PBS Buffer.

1.5 Tween-20.

1.6 Immunohistochemical oil pen.

1.7 Bond™ Dewax Solution. Cat:AR9222, Leica.

1.8 Bond™ Wash Solution 10 x concentrate. Cat: AR9590, Leica.

1.9 Bond™ Epitope Retrieval Solution 1. Cat: AR9961, Leica.

1.10 Bond™ Epitope Retrieval Solution 2. Cat: AR9640, Leica.

(Please choose the antigen repair solutions for antigen repair according to the Elabscience® Antibody manual)

1.11 Bond™ Polymer Refine Detection. Cat: DS9800, Leica.

1.12 Bond™ Titration kit: (The Bond Titration Kit contains Bond Titration Container Inserts and Bond Titration Containers)Hematoxylin Staining Solution. Cat: OPT9049, Leica.

1.13 Bond™ Aspirating Probe Cleaning. Cat: CS9100, Leica.

1.14 Bond™ Printer Ribbon & Labels Cxi (6Pack) Cat: S21.4610.A, Leica.

1.15 Bond™ Universal Covertiles. Cat: S21.2001, Leica.

1.16 Neutral Balsam.

2) Instruments

2.1 Automatic Immunohistochemical Staining System: BOND™ MAX, Leica.

3) Staining Procedure

Ensure the type and quantity of tissue sections and reagents, set the “Reagent Setting” step, the “Slide Setting” step, the “Program Setting” step and other related settings through the software, determine the staining operation procedure, print the label, paste the label on the corresponding tissue slices, put the labeled slices and reagents into the staining machine for quality inspection, and enter the staining after passing the quality inspection. The procedure is as follows:

3.1. Bake and Dewax Protocol

3.1.1 Heat the slice to 60°C and bake for 30 min.

3.1.2 Heat the slice to 72°C, add 150 µL Bond Dewax Solution on it and incubate for 30 s, then discard the solution.

3.1.3 Maintained at 72°C, add 150 µL Bond Dewax Solution to wash the slice, then discard the solution.

3.1.4 Cool the slice to RT, add 150 µL Bond Dewax Solution to wash the slice, then discard the solution.

3.1.5 Add 150 μL alcohol to wash the slice and discard the alcohol at RT, wash for 3 times.

3.1.6 Add 150 µL Bond Wash Solution and wash the liquid at RT, wash for twice.

3.1.7 Add 150 µL Bond Wash Solution and incubate for 5 min at RT, then discard the solution.

3.2. HIER 20 min with ER1/ER2 Protocol

3.2.1 Add 150 µL ER1 or ER2 to wash the slice at RT.

3.2.2 Add 150 µL ER1 or ER2 at RT then heat to 100°C and incubate for 20 min.

3.2.3 Cool the slice to RT, and incubate for 20 min.

3.2.4 Heat the slice to 35°C, add 150 µL Bond Wash Solution to wash the slice, then discard the solution.

3.2.5 Cool the slice to RT, add 150 µL Bond Wash Solution and incubate for 3 min, then discard the solution.

3.3. IHC Protocol

3.3.1 Add 150 µL Peroxide Block on the slice and incubate for 5 min at RT.

3.3.2 Add 150 µL Bond Wash Solution to wash the slice then discard the solution, wash for 3 times.

3.3.3 Add 150 µL primary antibody from Elabsciecne and incubate for 30 min at RT.

3.3.4 Add 150 µL Bond Wash Solution to wash the slice then discard the solution, wash for 3 times.

3.3.5 Add 150 µL Post Primary to the slice and incubate for 8 min at RT, then discard the Post primary.

3.3.6 Add 150 µL Bond Wash Solution to wash the slice then discard the solution, wash for 3 times.

3.3.7 Add 150 µL Polymer to the slice and incubate for 8 min at RT, then discard the Polymer.

3.3.8 Add 150 µL Bond Wash Solution to wash the slice then discard the solution, wash for twice.

3.3.9 Add 150 µL Deionized Water to wash the slice then discard the Deionized Water.

3.3.10 Add 150 µL Mixed DAB Refine to the slice and incubate for 10 min at RT, then discard the Mixed DAB Refine.

3.3.11 Add 150 µL Bond Wash Solution to wash the slice then discard the solution, wash for 3 times.

3.3.12 Add 150 µL Hematoxylin to the slice and incubate for 5 min at RT, then discard the Hematoxylin.

3.3.13 Add 150 µL Deionized Water to wash the slice.

3.3.14 Add 150 µL Bond Wash Solution to wash the slice.

3.3.15 Add 150 µL Deionized Water to wash the slice.

3.4. Dehydration and Transparency

3.4.1 At the end of the staining procedure, take the slice and discard the Deionized Water.

3.4.2 Immerse the slice in 75% ethanol for 30 s/time, 2 times

3.4.3 Immerse the slices in 95% ethanol for 30 s/time, 2 times.

3.4.4 Immerse the slices in absolute ethanol for 30 s/time, 2 times.

3.4.5 Immerse the slices in transparent dewaxing solution for 5 min

Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.

3.5. Seal the Slices

3.5.1 Take out the slices and drain them (about 5 s), blow them dry with cold air by blower.

3.5.2 Add 1~2 drops of neutral balsam to the slice (depending on the number and size of the slice), and cover the slide.

3.5.3 Check the slice to make sure that the slice tissue is completely covered with neutral balsam, and there is no big bubble in the tissue (discharge big bubble by squeezing the cover glass).

3.5.4 Place slices in fume hood to dry.

4). Observe the slice by microscope and judge the result.

4. Notices

1) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer BOND MAX with matched immunohistochemical staining system. Due to the different kinds / targets of Antibody for IHC, the antigen repair methods may differ, please read the manual carefully and strictly follow the manual.

2) This method recommends the varieties / Models / manufacturers of the important related reagents in the process of immunohistochemistry experiment. If users change the corresponding reagents / raw materials, you need to evaluate the equivalence of the alternative varieties and seek the optimal operating procedures.

3) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer BOND MAX with matched immunohistochemical staining system. If the user changes the immunohistochemical staining system, please evaluate the equivalence of the alternative system and find the optimal operation procedure.

1. Target

This method is a guide to use the Elabscience ® RTU antibody for IHC correctly with Benchmark XT (Roche) for immunohistochemical operation and obtain the optimal results.

2. Limits

This method is suitable for the automatic immunohistochemical staining experiment on the Benchmark XT (Roche) instrument with the Elabscience ® RTU antibody for IHC.

3. Experimental Steps

1) Reagents and Consumables

1.1 Transparent dewaxing solution.

1.2 Skim Milk Powder

1.3 Absolute ethanol.

1.4 95% ethanol.

1.5 10× EZ PREP SOLUTION. Cat: 05279771001, Roche.

1.6 Reaction Buffer Concentrate (10×). Cat: 05353955001, Roche.

1.7 LCS (Predilute). Cat: 05264839001, Roche.

1.8 10× SSC SOLUTION Cat: 05353947001, Roche.

1.9 CELL CONDITIONING SOLUTION, CC1 (pH 8.4). Cat: 05279801001, Roche.

1.10 UltraView Universal DAB Detection Kit. Cat: 05269806001, Roche.

1.11 Blue Return Solution: BLUING REAGENT. Cat: 05266769001, Roche.

1.12 Hematoxylin Staining Solution: HEMATOXYLIN II Cat: 05277965001, Roche.

1.13 KIT PACK, EBAR (US/EUROPE). Cat: 05248850001, Roche.

1.14 RIBBON, EBARPRINTER. Cat: 05250889001, Roche.

1.15 Neutral Balsam.

2) Instruments

2.1 Automatic Immunohistochemical Staining System: Benchmark XT, Roche.

3) Staining Procedure

3.1. Baking Slice

3.1.1 Select the target tissue slices to be examined, and mark the slices with correct name and markers.

3.1.2 Insert the slices into the dyeing frame and put it into the oven (62°C) to bake the slices for 1 h.

3.2. Print Labels

3.2.1 Click “Barcode Label” to write the label information, print the labels, and paste the labels on the slices.

3.3. Immerse the Slices in 10% Skim Milk Solution

3.3.1 Add 20 g skimmed milk powder into 200 ml distilled water and the mixture is 10% skimmed milk solution.

3.3.2 Immerse the labeled slices in 10% Skim Milk Solution for 15 min to avoid tissue exfoliation.

3.3.3 Wash the slices with tap water to wash most of the 10% skimmed milk solution and wait for the IHC staining.

3.4. Benchmark XT Staining Procedure

3.4.1 Place the slices on the slicing disk, dry the moisture on the slices, and ensure that each slice is placed stably, all of which are in the four snaps of the slicing disk.

3.4.2 Put DAB kit, Hematoxylin Staining Solution, Blue Return Solution and other reagents into the reagent rack (please make sure that the corresponding reagents are sufficient), and check whether the sampling port is full of liquid. If it is found that it is not full of liquid, please squeeze the reagent bottle, exhaust the air, and let the liquid fill the filling port to form a half moon liquid surface.

3.4.3 Set protocol through Ventana nexes software, click Run to complete the pre operation confirmation information. Click START RUN, and the system will conduct self-inspection first, then the program will start to run.

3.4.4 IHC Staining Program

3.4.4.1 Paraffin [Selected].

3.4.4.2 Deparaffinization [Selected].

3.4.4.3 Cell Conditioning [Selected].

3.4.4.4 Conditioner 1 [Selected].

3.4.4.5 Mild CC1 95Deg C and incubate for 30 min [Selected]. (After the completion of antigen repair, the instrument will prompt to pull out the slice table, add the primary antibody, then push the slice table into the instrument, press the prompt key on the instrument manually, and then enter the subsequent staining procedure.)

3.4.4.6 Ab Incubation Temperatures [Selected].

3.4.4.7 Disable Heat for Ab Inc. [Selected].

3.4.4.8 Titration [Selected].

3.4.4.9 Hand Apply (Primary Antibody), and Incubate for 32 min.

3.4.4.10 Counterstain [Selected].

3.4.4.11 Apply One Drop of [HEMATOXYLIN II] (Counterstain), Apply Coverslip, and Incubate for 12 min.

3.4.4.12 Post Counterstain [Selected].

3.4.4.13 Apply One Drop of [BLUING REAGENT] (Post Counterstain), Apply Coverslip, and Incubate for 4 min.

3.4.5 After the staining, take out the slice table, then take out the slices and wash with tap water containing detergent for cleaning. Ensure that the grease on each slice is cleaned, and then wash the slices with running water until they are cleaned, dry the residual water on the slices and enter the subsequent treatment.

3.5. Dehydration and Transparency

3.5.1 Immerse the slices in 75% ethanol for 30 s/time, 2 times.

3.5.2 Immerse the slices in 95% ethanol for 30 s/time, 2 times.

3.5.3 Immerse the slices in absolute ethanol for 30 s/time, 2 times.

3.5.4 Immerse the slices in transparent dewaxing solution for 5 min.

Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.

3.6. Seal the Slices

3.6.1 Take out the slices and drain them (about 5 s), blow them dry with cold air by blower.

3.6.2 Add 1~2 drops of neutral balsam to the slice (depending on the number and size of the slice), and cover the slide.

3.6.3 Check the slice to make sure that the slice tissue is completely covered with neutral balsam, and there is no big bubble in the tissue (discharge big bubble by squeezing the cover glass).

3.6.4 Place slices in fume hood to dry.

4). Observe the slice by microscope and judge the result.

4. Notices

1) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Benchmark XT with matched immunohistochemical staining system. Due to the different kinds / targets of Antibody for IHC, the antigen repair methods may differ, please read the manual carefully and strictly follow the manual.

2) This method recommends the varieties / Models / manufacturers of the important related reagents in the process of immunohistochemistry experiment. If users change the corresponding reagents / raw materials, you need to evaluate the equivalence of the alternative varieties and seek the optimal operating procedures.

3) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Benchmark XT with matched immunohistochemical staining system. If the user changes the immunohistochemical staining system, please evaluate the equivalence of the alternative system and find the optimal operation procedure.

1. Target

This method is a guide to use the Elabscience ® RTU antibody for IHC correctly with Autostainer link 48 (Dako) for immunohistochemical operation and obtain the optimal results.

2. Limits

This method is suitable for the automatic immunohistochemical staining experiment on the Autostainer link 48 (Dako) instrument with the Elabscience ® RTU antibody for IHC.

3. Experimental Steps

1) Reagents and Consumables

1.1 Transparent dewaxing solution.

1.2 Absolute ethanol.

1.3 95% ethanol.

1.4 PBS Buffer.

1.5 Tween-20.

1.6 Immunohistochemical oil pen.

1.7 EnVision™ TMFLEX+, High pH (Link) Cat: K800221-2CN, Dako.

1.8 DAB Away Cat: S196730-2, Dako.

1.9 User Fillable Reagent Bottle, 12 mL Capacity, (Link) Cat: SK20110- 2, Dako.

1.10 User Fillable Reagent Bottle, 5 mL Capacity, (Link) Cat: SK20005-2, Dako.

1.11 Large Flap Slide Label Kit (875×950) Cat: S341730, Dako.

1.12 Hematoxylin Staining Solution.

1.13 Neutral Balsam.

2) Instruments

2.1 Oven.

2.2 PT Link: PT200, Dako.

2.3 Automatic Immunohistochemical Staining System: Autostainer Link 48, Dako.

3) Staining Procedure

3.1 Baking Slice

3.1.1 Select the target tissue slices to be examined, and mark the slices with correct name and markers.

3.1.2 Insert the slices into the dyeing frame and put it into the oven (62°C) to bake the slices for 1 h.

3.2 Deparaffinizing and Rehydration

3.2.1 Immerse the slices in transparent dewaxing solution for 15 min/time, 2 times.

3.2.2 Immerse the slices in absolute ethanol for 5 min/time, 2 times.

3.2.3 Immerse the slices in 95% ethanol for 5 min/time, 2 times.

3.2.4 Immerse the slices in 75% ethanol for 5 min/time, 2 times.

Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.

3.2.5 Take out the dyeing frame with slices, Drain away the solution. Washing with water for 10 s/time, more than 3times.

3.3 Antigen Retrieval

3.3.1 According to the manual, choose EnVision™ FLEX Target Retrieval Solution High pH (50×), and repair the antigen with PT Link as below.

3.3.2 Take appropriate amount of antigen repair solution into the repair tank, preheat to 75°C in advance.

3.3.3 Immerse the dyeing frame with slices into the repair solution of the repair tank immediately, cover the tank, heat to 97°C, and incubate for 30 minutes.

3.3.4 Wait until the Retrieval Solution is cold about 75°C, uncover the tank, take out of the dyeing frame with slices, Immerse them in Wash Buffer to chill-down.

3.3.5 After the section is cooled, remove the liquid from the slices. Place the dyeing frame on the absorbent paper, draw a circle 2-3 mm away from the tissue with an immunohistochemical oil pen on each slice, then place the slices on the slice frame of Aautostainer link 48.

3.4 For the Autostainer Link 48 staining procedure, choose the FLEX program. The main parameters are as follows

3.4.1 FLEX Peroxidase Block (SM801), Volume (µl): 100, Incubation: 5 min.

3.4.2 Elabscience^® RTU Antibody for IHC, Volume (µl): 100, Incubation: 30 min.

3.4.3 FLEX /HRP (SM802), Volume (µl): 100, Incubation: 30 min.

3.4.4 FLEX DAB+ Sub-Chromo (SM803), Volume (µl): 200, Incubation: 10 min.

3.4.5 Refer to flex procedure for other dyeing procedure parameters.

3.4.6 Elabscience^® RTU Antibody for IHC, P40 Monoclonal Antibody (Cat: PA6665)

Reagents and Incubation

Staining Procedure

3.5 Hematoxylin stain the nucleus

3.5.1 After the Autostainer Link 48 staining , take the slices out of the slice frame and put them on the dyeing frame, wash with water for 10 s/time, 2~3 times, Remove the liquid from the dyeing frame, put them into the hematoxylin dyeing solution, and incubate for 7 min.

3.5.2 Take out the slices and drain them (about 5 s), wash them with water to remove the superfluous dye, put the slices into PBST for 60 s, then wash with water for 10 s/time, 3 times, then remove the water from the dyeing frame.

3.6 Dehydration and transparency

3.6.1 Immerse the slices in 75% ethanol for 30 s/time, 2 times.

3.6.2 Immerse the slices in 95% ethanol for 30 s/time, 2 times.

3.6.3 Immerse the slices in absolute ethanol for 30 s/time, 2 times.

3.6.4 Immerse the slices in transparent dewaxing solution for 5 min.

Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.

3.7 Seal the slices

3.7.1 Take out the slices and drain them (about 5 s), blow them dry with cold air by blower.

3.7.2 Add 1~2 drops of neutral balsam to the slice (depending on the number and size of the slice), and cover the slide.

3.7.3 Check the slice to make sure that the slice tissue is completely covered with neutral balsam, and there is no big bubble in the tissue (discharge big bubble by squeezing the cover glass).

3.7.4 Place slices in fume hood to dry.

4). Observe the slice by microscope and judge the result.

4. Notices

1) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Link 48 with matched immunohistochemical staining system. Due to the different kinds / targets of Antibody for IHC, the antigen repair methods may differ, please read the manual carefully and strictly follow the manual.

2) This method recommends the varieties / Models / manufacturers of the important related reagents in the process of immunohistochemistry experiment. If users change the corresponding reagents / raw materials, you need to evaluate the equivalence of the alternative varieties and seek the optimal operating procedures.

3) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Link 48 with matched immunohistochemical staining system. If the user changes the immunohistochemical staining system, please evaluate the equivalence of the alternative system and find the optimal operation procedure.

Deparaffinizing and rehydration

1. Immerse the slides inXyleneⅠand XyleneⅡsuccessively for 10 minutes respectively.

2. Immerse the slides in anhydrous ethanolⅠ,anhydrous ethanolⅡ, 95%ethanol, 80% ethanol, and 70% ethanol successivelyfor 5 minutes respectively, and then use deionized water to wash the slidesfor 2 minutes and repeat 2 times.

Antigen retrieval (optional)

3. Put the slides into the repair box, and then add 0.01M citric acid buffer (PH6.0) to make the tissue immersed.

4. Repair the antigen with medium power microwave for 10 minutes (start timing when the liquid boils), and avoid the tissue from drying during the process.

5. Take out the repair box from microwave oven, and it cool down naturally. Take out the slides when the repair solution cooled down to room temperature, and wash the slides with PBS (PH7.4) for 3 minutes and repeat 3 times. (Don’t flush the tissue directly during the washing process in order to avoid breaking up the tissue).

Inactivation

6. Add the prepared 3% H2O2to the slides drop by drop to block endogenous peroxidase. Then incubate them at room temperature for 15 minutes (dilute 30% H2O2with methanol or distilled water), finally use PBS to wash the slides with PBS for 3 minutes and repeat 3 times.

Antibody incubation

7. Blot up absorbent paper with PBS, and add 5% normal serum (Sharing the same or similar species with secondary antibodies) drop by drop on the sections, then block it at 37℃for 30 minutes.

8. Wipe dry the liquid around the tissue on the slides with absorbent paper, and use an oil pen to draw a circle around the tissue, and then add the diluted primary antibodies drop by drop. Add PBS to the section of controls if the negative controls are required. After adding primary antibodies, put the slides into wet box to be incubated at 4°C overnight. (The optimum dilution ratio of the antibodies should be pre-determined through experiments in advance)

9. Wash the slides with PBS for 3 times, each time for 2 minutes, and then add HRP-conjugated secondary antibodies after wiping dry the slides with absorbent paper, finally incubate the slides at 37℃for 30 minutes.

Signal detection

10. Wash the sections with PBS for 3 minutes and repeat 4 times, and wipe dry the sections with absorbent paper, then add DAB substrate reagent that is prepared freshly drop by drop to each section, and observe them under a microscope. The positive signal appears brown-yellow or brown in color. The time should be well controlled to avoid the color appears too deep. Wash the section with tap water to terminate the reaction.

11. Hematoxylin counterstaining: Immerse slides in Harris hematoxylin solution for about 30 seconds to 1 minute, and then transfer slides into ethanol solution with 1% HCl after washing with water, finally wash them with water. (Optional)

Dehydration and mounting

12. Firstly immerse the slides in water and wash them, and then put the slides into the following reagents in order to dehydrate and permeate: 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanolⅠ,anhydrous ethanol,XyleneⅠand XyleneⅡ. Put the slides in each reagent for 2 minutes, and finally air dry the sections in the fume cupboard.

13. Drop resinene beside the section, and then cover them with the cover glass. In order to avoid air bubble, firstly lay one side of the cover glass flat and then gently lay another side flat. Finally dry the sealed sections by laying them in thefume cupboard

14. Observe the dried sections and collect images with a microscope.

Immunohistochemistry (IHC) is a technology applying the antigen-antibody specific binding principle to determine the position, qualitative and quantitative properties of intracellular antigen (polypeptide and protein) through the colorimetric chemical reaction of the labelled antibody color reagent (fluorescein, enzyme, metal ion, isotope).

Immunohistochemistry is not difficult, but it’s not easy to make beautiful dyeing results. The key to success is that immunohistochemistry detail decides success or failure. what is the “details”? Here are the details and process. (Paraffin tissue section as an example)

1.Specimen fixation

The purpose of fixation is not only to solidify protein in cells, to reduce or terminate the reaction of endogenous or exogenous intracellular decomposing enzymes, but also to prevent autolysis of tissue cells, so as to preserve the antigenicity of tissues or cells, so that antigens do not lose activity and or disperse. The tolerance of different antigens to the fixed solution is different, so the appropriate fixative should be chosen according to the antigens.

Now the commonly used fixers are neutral formaldehyde, 4% polyformaldehyde – phosphate buffer. Some fixed agents have better effect on special organization, for example, picric acid has a fixed effect for softening scalp and nails, Helly is better for fixing islet and pituitary, PLP is better for fixing liquid sugar and preserved tissue antigen.

2. Dehydration, paraffin embedded and cut into slices

Dehydration with gradient ethanol (low to high) and full dehydration, for some brittle tissues, such as liver, spleen, etc. It should reduce the retention time of high concentration alcohol, and transparent time should also be controlled.

For tissue impregnation, the paraffin wax is usually used at 56 ℃~58℃ melting point, and the best temperature of the wax impregnation is not more than 60 ℃, so as to prevent the loss of antigen.

Buried the tissue quickly so that the section has a complete cut. Check if the blade is notched in time to prevent the waxes from scratching.

3.Deparaffinizing and rehydration

Deparaffinize the section to the normal state and exposes the antigen to facilitate the binding with the antibody. If the dewaxing or rehydration not prone to focal reaction and immersion is not complete, it may cause nonspecific background staining.

4.Antigen retrieval

Since the tissues are immobilized in formaldehyde or polyformaldehyde, the crosslinking of proteins and the sealing of aldehyde groups have been taken to cover the antigenic determinants. Through the antigen retrieval, the antigen determination re-exposed to make the antigen be detected by specificity antibodies.

The commonly retrieval methods are divided into three types from strong to weak, high pressure heating repair, microwave repair, and pancreatin repair. High pressure heating repair is simple and easy to operate, and the effect is better than the others.

It is very important to control the retrieval temperature and time when use high pressure heating. The higher temperature, the shorter of repair time, the temperature is negatively related to the repair time. After the retrievalling, cooling the section at room temperature, so that the protein refolds naturally. Use excessive antigen repair solution to prevent the high temperature liquid volatilization dry to avoid causing irreversible damage to the slices.

5.Inactivation

In the traditional ABC method and SP method, the immunohistochemical reaction is easily interfered by endogenous peroxidase and biotin, so the slices must be inactivated and blocked by hydrogen peroxide and ovalbumin.

It’s generally inactivated endogenous peroxidase with 3% hydrogen peroxide for 10 min, and the use of methanol to dilute hydrogen peroxide is better for protecting antigens and fixed tissues.

6.Serum blocking

In order to prevent the non-specific combination of the primary antibody with the tissue, the BSA or goat serum can be used to block these loci. The blocking serum is usually from the same animal of secondary antibody and the blocking time is 10-30 min at room temperature.

7.Antibody incubation

Different antibody concentration, incubation time and temperature have a great influence on the dyeing results. At 4℃, the reaction is slow and the background is shallow. The reaction is faster at 37 ℃ so the incubation time must be shorter. Choosing room temperature incubation is helpful for the convenience of the experimental process, and it also applies to the detection of more samples. It’s general to incubate secondary antibody at room temperature for 30 min.

8.slices wishing

In order to prevent nonspecific staining caused by residues such as primary antibody and secondary antibody, proper wishing is especially important. It is recommended to wash 30s for 3 times, and TBST can be used separately after primary antibody incubation. While wishing, we should pay attention to prevent the contamination from the cross reaction, gently rinse and prevent the removal of the tablet. The pH of TBS is suggested to be used as 7.6-8.0, with a concentration of 0.01 M.

9.DAB Developing

The background and the depth of the specific staining can be determined by the conditions of DAB incubation. The color time of DAB is not fixed, and the time is mainly controlled under the microscope, washing the slices when the specific color is stronger and the background color is shallow.

DAB incubate for a short time (a few seconds or ten seconds) appears with deep brown, it means that he antibody concentration is too high, need to dilute the antibody concentration; Deep background with DAB incubate for a short time, there may be nonspecific protein insufficiency, need to extend the blocking time; DAB incubate for a long time (more than ten minutes) to appear positive staining may be caused by low concentration of antibody or too long time with blocking time.

For a weaker DAB color, an enhancement method can be taken sometimes. Adding metal ions such as copper sulfate, gomori methenamine silver, cobalt chloride, nickel sulfate, nickel chloride and imidazole.

10.Re-dyeing

After immunohistochemical staining, the cell nucleus must be stained to foil the structure of the tissue. At present, Mayer’s hematoxylin is commonly used for dyeing about 2 min. It can shorten the re-dyeing time when nucleoprotein is dyed by DAB, then ammonia or pH 8 TBS returns to blue.

11.Dehydration and mounting

In order to preserve the Immunohistochemistry result for a long time, neutral balsam was used as mounting. Prevent effects of bubble with glass mounting a. If the neutral balsam is sticky, xylene can be added to dilute to the neutral balsam which makes neutral balsam quickly spread while mounting. It’s recommended that mounting while there is still xylene residual and operates in the fuming cupboard which helps to remove the bubble clean and does not affect the follow-up mirror inspection.