All-in-One Kit for Immunohistochemical Staining for Tissues with Antibodies

1. Specific antigen on the tissue binds to unlabeled primary antibody.


2. Labeled secondary antibody conjugated to an enzyme (HRP) binds to the antigen-antibody complex, thus amplifying the signal greater than direct staining methods.


3. Enzyme (HRP) forms a colored (brown), insoluble precipitate at the antigenic sites in the presence of a substrate (H₂O₂) using a chromogen, DAB (3, 3'-diaminobenzidine) that is easily visualized with light microscopy.  IHC staining is simple and easy to perform at the benchtop without the need for any specific instrumentation and allows you to detect antigens in context of tissue morphology. The colored precipitate from DAB is stable for several years providing a permanent record.

• SuperPicTure™ Polymer Detection Kit for highly sensitive and specific immunohistochemical staining of tissue samples using HRP.
• Peroxo-Block™, a specific inhibitor of endogenous peroxidase activity, to effectively eliminate endogenous peroxidase activity without interfering with specific immunostaining.
• Antibody Diluent for diluting primary antibodies. 
• PBS Powder and 50% Tween-20 to prepare washing buffer.
• Mini PAP pen draws a water repellent circle around slide mounted tissue, preventing any wasting of valuable reagents by keeping the liquid pooled in a single droplet.
• Hematoxylin Counterstain Reagent for brilliant staining of nuclei without interfering with the chromogen signal.
• Histomount™, an organic based mounting medium, for immunohistochemical procedures. 

An experimental workflow for using the BioModule™ IHC Staining Unit with formalin-fixed paraffin embedded tissues is shown below.




Materials Needed
 
Materials supplied with the BioModule™ IHC Staining Unit for Tissues and User Supplied materials are listed below. Ordering information is below

.

• A specimen processed in the same way as the unknown and contains the antigen to be stained.

• An additional slide that is treated with a non-immune serum or isotype control antibody that matches the isotype of the experimental antibody instead of the same concentration of primary antibody. Any staining observed on the specimen is probably due to non-specific protein binding or non-specific binding of other reagents.
• Rabbit and mouse primary antibody isotype controls are available from Invitrogen.

• A specimen processed in the same way as the unknown but does not contain the antigen to be stained (optional).  

• Fresh, frozen tissue, or paraffin embedded tissue of choice
• Microtome and cryostat for tissue sectioning
• Pre-coated slides (see below)
• Coplin jars or equivalent
• PBS (supplied with the kit)

• OCT® Compound
• Chilled 100% acetone for fixing

• Xylene
• Graded series of ethanol (80%, 95% and 100% ethanol)
• 55ºC oven

1. Chill 100% acetone at 4ºC (required for Step 7, below).


2. Obtain fresh tissue.


3. Snap freeze fresh tissues in cryomolds containing OCT® (Optimal Cutting Temperature) compound (a solution of glycols and resins which provides an inert matrix for sectioning). Store frozen tissue blocks at -70ºC until you are ready for tissue sectioning.


4. For sectioning, allow the frozen tissue block to equilibrate to the cryostat temperature.


5. Cut 4-6 µm cryostat sections and mount on coated glass slides.


6. Dry tissue sections at room temperature for 30 minutes. If desired, store slides at -70ºC before fixing. If slides are stored at -70ºC, warm the slides to room temperature before the fixing step.


7. Place the slides in 100% acetone at 4ºC for 10 minutes to fix the sections.


8. Remove slides from acetone and air dry for 10-30 minutes.


9. Circle each tissue section using the Mini PAP Pen.


10. Store at -70ºC until use or wash the slide in PBS for 10 minutes and proceed immediately to Peroxidase Quenching.

1. Obtain or prepare the formalin-fixed paraffin embedded sections of choice.


2. Dry slides containing 4 µm formalin-fixed paraffin embedded sections in a 55ºC oven for 2 hours or overnight (do not allow the temperature to exceed 60ºC). Store the slides containing the formalin-fixed paraffin embedded tissue sections at room temperature until needed.


3. Place slides in xylene for 5 minutes at room temperature.


4. Remove slides and place in xylene a second time for an additional 5 minutes.


5. Remove slides and place in 100% ethanol 2 times for 5 minutes each time.


6. Remove slides and place in 95% ethanol for 5 minutes.


7. Remove slides and place in 80% ethanol for 5 minutes.


8. Remove slides and place in PBS for 10 minutes.


9. Drain any excess reagent by tapping the edge of the slide on paper towels and wipe the area near the tissue sections with a laboratory wipe.


10. Circle each tissue section using the Mini PAP Pen.


11. Proceed immediately to Peroxidase Quenching.  

1. Perform peroxidase quenching step using the Peroxo-Block™, if your sample exhibits high endogenous peroxidase activity.


2. Incubate the samples with an appropriate dilution of the primary antibody.


3. Wash any unbound antibody 


4. Incubate the samples with HRP Polymer Conjugate.


5. Wash any unbound polymer conjugate.


6. Add diluted DAB chromogen to develop the signal. 


7. Perform counterstaining with hematoxylin.


8.  Mount the slides using Histomount™.


9. Visualize the staining using a light microscope.

• Perform all steps at room temperature, unless a different temperature is specified.
• Always wear protective clothing, safety glasses, and gloves while handling the slides and reagents.
• Do not allow the slides to dry once the staining procedure has started.
• To avoid dislodging the tissue cores, handle the slides gently, especially during the washing steps.
• Perform the staining procedure using Coplin jars, staining dishes or automated instruments.
• Perform all incubation steps with various solutions in a humidified chamber.
• Be sure to run appropriate controls.
• Determine the optimal titer of the antibody through serial dilutions. •Apply enough reagents to cover the tissue during reagent incubation steps; usually ~100-200 µl.
• Drain any excess reagents after washing by tapping the edge of the slide gently on the jar or on paper towels for a few seconds.

• Tissue sample
  
• Primary antibody
• Distilled water
• Xylene or xylene substitutes
• Ethanol (95% and 100%)
• Coplin jars or equivalent
• Humidified chamber
• Coverslips
• Optional: 3% hydrogen peroxide in methanol

• Hot plate
• 20X Citrate buffer, pH 6.0 (page 26) •20 X EDTA Solution, pH 8.0 (page 26, for antibodies that require EDTA instead of citrate buffer)
• Optional: Digest-All™ Kit for enzymatic digestion

• Peroxo-Block™
• SuperPicTure™ Polymer Detection Kit
• PBS and 50% Tween-20
• Antibody Diluent
• Hematoxylin Counterstain Reagent
• Histomount™
• Mini PAP Pen 

1. Treat frozen tissue slides (Step 9) with 100-200 µl Peroxo-Block™ or enough to cover the tissue section for 45 seconds. Do not incubate for more than 45 seconds.


2. Wash slides 3 times with 1X PBS for 2 minutes, each time.


3. Proceed immediately to Incubating with Primary Antibody. 

1. Perform this epitope retrieval protocol after Peroxidase Quenching   Note: You may use citrate buffer  or EDTA solution  depending on the antibody manufacturer’s recommendation.


2. Based on the reagent that you are using, dilute the reagent to 1X as follows:

To 25 ml 20X Citrate Buffer, pH 6.0 solution, add 475 ml distilled water to obtain a 1X Citrate Buffer, pH 6.0 solution.
 
To 25 ml 20X EDTA solution, pH 8.0, add 475 ml distilled water to obtain a 1X EDTA solution, pH 8.0.
 

3. Place the slides in a slide rack and place the rack in a 1 L glass beaker containing 500 ml diluted citrate buffer (1X), pH 6.0 or diluted EDTA (1X) solution, pH 8.0.


4. Place the beaker with slides on a hot plate. Heat the solution until it boils and continue boiling the solution for 15 minutes.
   


5. Remove beaker from the hot plate and allow the contents to cool for 25 minutes at room temperature.


6. Rinse slides with 1X PBS and proceed immediately to Incubation with Primary Antibody. 
   
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   Epitope Retrieval Protocol Using Enzymatic Digestion
   
   Brief protocol using Digest-All™ Kit is described below. The Digest-All™ is a flexible and standardized system for tissue digestion and contains trypsin, ficin, and pepsin proteolytic enzymes. The degree of digestion is based on a standardized 10 minute incubation at 37°C. The trypsin solution is supplied with diluent so that different trypsin concentrations can be made to generate a range of digestion activity between that of Ficin and Pepsin. 
   
   
   1. Perform this epitope retrieval protocol after Peroxidase Quenching.
   
   
   2. Add the digestion enzyme of choice to your tissue sections  Ficin and pepsin may be applied directly from the bottle. Trypsin requires dilution: 1 drop Trypsin concentrate to 3 drops Trypsin Diluent. Mix well and apply to tissue sections.
   
   
   3. Incubate for 10 minutes at 37°C.
   
   
   4. Wash slides in several changes of 1X PBS and proceed immediately to Incubation with Primary Antibody.
      
      Incubation with Primary Antibody
      
      For best results, primary antibody dilution and incubation times need to be determined empirically and are dependent on sample preparation, antibody affinity, amount of antigen present, and antigen accessibility. Refer to the Primary Antibody section on page 10 for more details.
      The HRP Polymer Conjugate will react with mouse, rabbit, guinea pig, and rat primary antibodies.
       
      
      1. Dilute primary antibody in Antibody Diluent as recommended by the antibody manufacturer or as determined by a titration experiment. Alternatively, you may use prediluted 2nd Gen primary antibodies.
      
      
      2. Apply 100 µl or enough of the diluted primary antibody to completely cover the tissue on each section.
      
      
      3. Incubate the slides in a humidified chamber for 30 minutes.
      
      
      4. Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.
      
      
      5. Proceed immediately to Incubation with HRP Polymer Conjugate, below
       
      Incubation with HRP Polymer Conjugate
      
      
      1. Apply 2 drops (100 µl) or enough of the ready-to-use HRP Polymer Conjugate (Reagent A) to completely cover the tissue on each section.
      
      
      2. Incubate the slides in a humidified chamber for 10 minutes.
      
      
      3. Wash slides 3 times with 1X PBS containing 0.05% Tween-20 for 2 minutes, each time.
      
      
      4. Proceed immediately to Signal Development, below.
       
      Signal Development
      
      
      1. Dilute the 20X DAB Chromogen supplied with the kit in a sterile microcentrifuge tube just prior to use as follows:
      
      Distilled water                                              1 ml
      Reagent B1                                                  1 drop
      Reagent B2                                                  1 drop
      Reagent B3                                                  1 drop
      
      Mix well. Protect from light and use within 1 hour.
       
      
      2. Apply 2 drops (100 µl) or enough of the diluted DAB chromogen (prepared in Step 1) to completely cover the tissue on each section.
       
      Caution: DAB is a known carcinogen; handle with care.
       
      Incubate the slides in a humidified chamber for 5 minutes or until the desired signal intensity is reached.
      Incubation time can vary depending on the antibody.
       
       Rinse thoroughly with distilled water.
       
       Proceed immediately to Counterstaining, below.
      
      Counterstaining
      
      
      1. Apply 2 drops (100 µl) or enough of the Hematoxylin Counterstain Reagent to completely cover the tissue on each section.
      
      
      2. Incubate the slides in a humidified chamber for 1-3 minutes.
      
      
      3. Rinse thoroughly with distilled water.
      
      
      4. Dip the slide in ammonia water 1X PBS or (0.25% NH3) to blue the nuclei.
      
      
      5. Rinse thoroughly with distilled water.
      
      
      6. Dehydrate through increasing concentrations of ethanol (95%,100%) for 10-20 dips each. Clear in 2 changes of xylene or xylene substitutes for 10-20 dips each.
      
      
      7. Proceed immediately to Mounting, below.
       
      Mounting
      
        1.     Apply 2-4 drops (100-200 µl) of Histomount™ mounting medium to the slide.
       
        2.     Apply a cover slip on the slide.
       
        3.     Allow the medium to dry at room temperature overnight.
       
      Microscopy
      
      Evaluate the results by examining the slides using a light microscope at 20x magnification. Interpret the results as described below.
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Example of Results
 
An example of results obtained after performing an immunohistochemical staining experiment with the BioModule™ Immunohistochemistry (IHC) Staining for Tissue is shown below.
 
In this experiment, formalin-fixed paraffin-embedded human testis tissue was subjected to the immunohistochemical staining protocol using 2ndGen predilute (ready-to-use) rabbit anti-phospho-MAP kinase ERK1/2 antibody from Invitrogen (cat. no. 08-1389)as described in this unit manual. 
The image shows phospho-MAP kinase-positive cells that are stained brown from the DAB chromogen, displaying the expected nuclear staining pattern.

Introduction

Review the information in this section to troubleshoot your immunohistochemical staining experiments.