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Reagents

Immunohistochemistry

The Immunohistochemistry staff provides the specifications for solution preparation and the contact information for the companies who supply the reagents.

0.3% Hydrogen Peroxide

Hydrogen peroxide is used to quench endogenous peroxidase in immunohistochemical staining. A 0.3% solution is used on frozen tissue sections.

Prepare 200 ml of 0.3% hydrogen peroxide by mixing 2 ml of 30% hydrogen peroxide with 198 ml of deionized water.

Supplies:

30% Hydrogen Peroxide
Thermo Fisher Scientific
www.fishersci.com
Catalog No. H325


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Lectin Diluent

This solution (1mM CaCl2⋅2H2O, MgCl2, MnCl2) is the diluent for lectin reagents used in immunostaining.

 

To prepare this diluent, mix 1.0 ml of MgCl2 and 1.0 ml of MnCl2 in 900 ml of 1X Wash Buffer. Dissolve 0.147 g of CaCl2⋅2H2O into this solution using the stir plate. Add 0.5 ml of Tween 20 (0.05%) to the solution. Adjust the pH to 7.4. Then bring the final volume up to 1.0 L. This diluent is stable at room temperature.

 

Supplies:

 

Calcium Chloride Dihydrate (CaCl2⋅2H2O)
Avantor Performance Materials
www.avantormaterials.com
Center Valley, PA 18034
610-573-2600
Catalog No. 4160

 

Magnesium Chloride Solution
Sigma-Aldrich
www.sigmaaldrich.com
St. Louis, MO 63178
866-266-2015
Catalog No. M1028

 

Manganese (II) Chloride Solution
Sigma-Aldrich
www.sigmaaldrich.com
St. Louis, MO 63178
866-266-2015
Catalog No. M1787

 

Tween 20
Bio-Rad Laboratories
www.bio-rad.com
Hercules, CA 94547
800-424-6723
Catalog No. 170-6531


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ProLong® Gold Mounting Media

ProLong® Gold reagent is an antifade liquid mountant used for coverslipping fluorescently labeled cell or tissue samples on microscope slides. This mounting media can be purchased with or without DAPI. DAPI (4',6-diamidino-2-phenylindole) can ne thought of as a counterstain that labels the nuclei in a sample.

 

Supplies:

 

ProLong® Gold Antifade Reagent Life Technologies / Invitrogen
Grand Island, NY 14072
www.invitrogen.com
888-584-8929
Catalog No. P36934

 

ProLong® Gold Antifade Reagent With DAPI Life Technologies / Invitrogen
Grand Island, NY 14072
www.invitrogen.com
888-584-8929
Catalog No. P36935


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1X Wash Buffer

Dako Wash Buffer is supplied as a 1-L concentrated Tris-buffered saline solution (10X) containing Tween 20, pH 7.6 (±0.1). It is sufficient for preparing 10 L of working solution.

 

Prepare 1X Wash Buffer by mixing the 1-L 10X concentrate with 9 L of deionized water.

 

Supplies:

 

Dako Wash Buffer 10X
Dako North America, Inc
www.dakousa.com
Carpinteria, CA 93013
800-235-5763
Code S3006


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3% Hydrogen Peroxide

Hydrogen peroxide is used to quench endogenous peroxidase in immunohistochemical staining. A 3% solution is used on formalin-fixed, paraffin-embedded tissue sections.

 

Prepare 200 ml of 3% hydrogen peroxide by mixing 20 ml of 30% hydrogen peroxide with 180 ml of deionized water.

 

Supplies:

 

30% Hydrogen Peroxide
Thermo Fisher Scientific
www.fishersci.com
Catalog No. H325

1% BSA Diluent

BSA diluent (1%) is used in preparing many of the reagents for immunohistochemiceal staining.

 

Prepare 500 ml by mixing 5 g of bovine serum albumin (BSA) with 500 ml of 1X Wash Buffer. Use a magnetic stir bar and stir plate to ensure that the powder is complete dissolved.

 

Supplies:

 

Bovine Serum Albumin (IgG-Free, Protease-Free)
Jackson ImmunoResearch
West Grove, PA
www.jacksonimmuno.com
800-367-5296
Catalog No. 001-000-162

 

Dako Wash Buffer 10X
Dako North America, Inc
www.dakousa.com
Carpinteria, CA 93013
800-235-5763
Code S3006


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DAB Chromogen

The components needed to prepare the DAB reagent are provided in the Dako Liquid DAB+ Substrate-Chromogen System. Add 1 drop of DAB chromogen to 1 ml of the substrate buffer and mix. Store the reagent in the dark until it is ready to be used.

 

Procedure:

 

  1. Wipe excess buffer from around the tissue section.
  2. Apply DAB reagent to tissues and incubate 6 minutes at room temperature in the dark.
  3. Rinse slides in running tap water for 3 min.
  4. Proceed with counterstaining step.

 

Results: Antigenic sites should appear brown.

 

Supplies:

 

Dako Liquid DAB+ Substrate-Chromogen System
Dako North America, Inc
www.dako.com
Carpinteria, CA 93013
800-235-5763 C
ode K3468


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1X Citrate Buffer

Citrate buffer is a pH 6.0 solution used in heat-induced epitope retrieval procedures.


Dilute the Antigen Decloaker 10X or Rodent Decloaker 10X down to a 1X concentration using deionized water. For instance, to prepare 10 L of working retrieval solution, dilute a 500-ml concentrate volume with 9.5 L of water.

 

Supplies:

 

Antigen Decloaker, 10X 
Biocare Medical 
www.biocare.net 
Concord, CA 94520 
800-799-9499 
Catalog No. CB910M

 

Rodent Decloaker, 10X 
Biocare Medical 
www.biocare.net 
Concord, CA 94520 
800-799-9499 
Catalog No. RD913


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Boric Acid-Borate Buffer

Stock Solution A: 1.24 g of boric acid in 100 ml deionized water
Stock Solution B: 1.9 g of sodium borate in 100 ml deionized water

 

Mix 85 ml of solution A with 15 ml of solution B. Adjust the volumes proportionately to make the amount you need. This solution is stable at room temperature.

 

Supplies:

 

Boric Acid (H3BO3)
Avantor Performance Materials
www.avantormaterials.com
Center Valley, PA 18034
610-573-2600
Catalog No. 2549

 

Sodium Borate (Na2B4O7.10H2O)
Avantor Performance Materials
www.avantormaterials.com
Center Valley, PA 18034
610-573-2600
Catalog No. 3570


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0.05M TRIS-HCl

The 0.05M Tris-HCl solution is the diluent used for trypsin during enzymatic epitope retrieval.

 

Prepare this reagent by mixing 6.057 g of TRIS, 1 g of CaCl2⋅2H2O, and 800ml of deionized water. Use a magnetic stir bar and stir plate to ensure that the powder is complete dissolved. Adjust the solution to pH 7.8 using 1N HCl. Then bring the final volume up to 1 L with deionized water. This solution is stable at room temperature for one month.

 

Supplies:

 

TRIS, Ultra Pure (C4H11NO3 MP Biomedicals Life Sciences
www.mpbio.com
Santa Ana, CA 92707
800-854-0530
Catalog No. 04819620

 

Calcium Chloride Dihydrate (CaCl2⋅2H2O)
Avantor Performance Materials
www.avantormaterials.com C
enter Valley, PA 18034 610-573-2600
Catalog No. 4160


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Normal Goat IgG - Affinity Purified

A purified normal goat IgG should be used as the negative control reagent when performing immunohistochemical staining with a purified goat IgG primary antibody. The amount of IgG in this reagent should be matched to that of the primary antibody and then applied to the samples that will serve as negatives.

Suppliers:

Normal Goat IgG
Santa Cruz Biotechnology, Inc.
Santa Cruz, CA 95060
www.scbt.com
1-800-457-3801
Catalog No. sc-2028

ChromPure Goat IgG, whole molecule
Jackson Immunoresearch Laboratories, Inc.
West Grove, PA 19390
www.jacksonimmuno.com
1-800-367-5296
Catalog No. 005-000-003

2N HCl

Prepare 1 L of 2N HCl by mixing 834 ml of deionized water and 166 ml of concentrated 12N HCl. This solution is stable at room temperature.

 

Caution: This solution should be prepared under a hood with the HCl slowly being added to the deionized water.

 

Supplies:

 

Hydrochloric Acid (HCl) (37%) Avantor Performance Materials
www.avantormaterials.com
Center Valley, PA 18034
610-573-2600
Catalog No. H613


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PLP Fixative

 

Part A - Making stock solutions. (All soln. for part A are made the same day as part B, which is one day prior to Part C)

 

  1. 8% Paraformaldehyde
    1. Weigh out 16g of Paraformaldehyde
    2. Measure out 200 mL of distilled water
    3. Add 160mL of the distilled water to the 16g paraformaldehyde
    4. Allow mixture to stir for at least 2hr at 37-50°
    5. After time has elapsed, add dropwise 10 N NaOH to clear soln.
    6. Slowly add remaining 40mL of water.
    7. Filter soln. and store in a dark bottle at 4°C
  2. 2M lysine-HCL prepared by dissolving 36.5g of lysine-HCl in 1L of deionized water
  3. 0.1M anhydrous dibasic sodium phosphate prepared by dissolving 14.2g of Na2HPO4 in 1L of deionized water
  4. 0.1M monobasic sodium phosphate (not anhydrous) prepared by dissolving 6.9g of NaH2PO4 in 500mL deionized water

 

Part B - Making the fixative

 

  1. Take 250ml of Lysine-HCl (#2) and use the dibasic solution (#3) to bring the lysine-HCl solution to a pH=7.4.
  2. Take 250ml of the dibasic soln (#3) and use the monobasic solution (#4) to bring the dibasic solution to a pH=7.4
  3. Take solution #5 and add enough of solution #6 to bring the volume to 500mL
  4. Repeat steps 5, 6, and 7 and combine the solutions for a total of 1000mL
  5. Store mixture at 4°C

 

Part C - Day of use.

 

  1. Use 600ml of solution lysine-phosphate solution (#8) and add 200mL of the paraformaldehyde solution (#1); allow to stir
  2. Add 1.712g of sodium periodate right before use. (Standard measurement .214g per 100 mL of fixative)


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4% Paraformaldehye (PF) Fixative

The procedure below makes approximately 100 ml of 4% paraformaldehyde fixative.

 

  1. Dissolve 4 g. of paraformaldehyde in 90 ml. of 1X PBS
  2. Heat gently to 58-60°C under a hood. Do not heat over 60°C (PF dissociates @ 60°C)
  3. Add 10N NaOH to clear the solution (pH 10 dissolves the paraformaldehyde). Usually 5-10 drops
  4. Remove from heat and pH
  5. Carefully pH to pH 7.0-7.5
  6. Bring to volume (100 ml) PBS (for a final concentration of 4 g. in 100 ml. of 1x PBS)
  7. Filter sterilize through a .22 micron filter
  8. Use at 4°C for RNA work. Can use at room temperature for non-RNA work

 

Note: Always prepare this fixative fresh.


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2% Paraformaldehye (PF) Fixative

The procedure below makes approximately 100 ml of 2% paraformaldehyde fixative.

 

  1. Dissolve 2 g. of paraformaldehyde in 90 ml. of 1X PBS
  2. Heat gently to 58-60°C under a hood. Do not heat over 60°C (PF dissociates @ 60°C)
  3. Add 10N NaOH to clear the solution (pH 10 dissolves the paraformaldehyde). Usually 5-10 drops
  4. Remove from heat and pH
  5. Carefully pH to pH 7.0-7.5
  6. Bring to volume (100 ml) PBS (for a final concentration of 4 g. in 100 ml. of 1x PBS)
  7. Filter sterilize through a .22 micron filter

 

Note: Always prepare this fixative fresh.


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1X EDTA

EDTA is a pH 8.5 solution used in heat-induced epitope retrieval procedures.

 

To prepare 1X EDTA, dilute 1 part concentrated EDTA Decloaker into 4 parts deionized water. This retrieval solution should be made up prior to each use.

 

Note: The solution is blue at room temperature. If it turns greenish-blue, green or yellow upon heating, the pH of the solution is not optimum; and the tissue staining may be significantly reduced. The grade of the water is also very important. If high-grade water is difficult to obtain, sterile water will need to be purchased.

 

Supplies:

 

EDTA Decloaker, 5X

Biocare Medical
www.biocare.net
Concord, CA 94520
800-799-9499
Catalog No. CB917


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Decolorizing Solution

This solution is used to remove hematoxylin from immunostains.

 

To prepare the decolorizing solution, mix 200 ml of 70% ethanol with 500 μL of concentrated HCl. Then proceed with the following steps:

 

  1. Submerge slides in solution for 2 minutes
  2. Rinse slides with deionized water
  3. Rinse slides in 1X wash buffer

 

Supplies:

 

100% Ethanol
Leica Biosystems
www.leica-microsystems.com
Buffalo Grove, IL 60089
800-225-3035
Catalog No. 3803686

 

Hydrochloric Acid (HCl) (37%)
Avantor Performance Materials
www.avantormaterials.com
Center Valley, PA 18034
610-573-2600
Catalog No. H613


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Hematoxylin

Hematoxylin is a reagent commonly used in biological staining. We use Modified Harris Hematoxylin as the counterstain for our immunohistochemical and immunocytochemical staining. It should be filtered daily to minimize the collection of crystals.

 

Supplies:

 

Modified Harris Hematoxylin 72711
Richard-Allan Scientific (Part of Thermo Scientific)
thermoscientific.com
Catalog No. 72711


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Carezyme II: Pepsin

Pepsin is a commonly used enzyme for proteolytic-induced enzymatic retrieval (PIER) in immunohistochemistry. The Carezyme II: Pepsin reagent is a ready-to-use pepsin solution used for the enzymatic pretreatment of formalin-fixed, paraffin-embedded tissues. The datasheet for this product has protocol recommendations. However, the end user should ultimately determine optimal working conditions (i.e., incubation times and temperatures) for their immunohistochemical staining.

 

Supplies:

 

Carezyme II: Pepsin
Biocare Medical
www.biocare.net
Catalog No. PEP956


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1% Dry Milk

Dry milk is sometimes used to block unwanted non-specific staining in immunohistochemistry. Equal parts of 1% dry milk (dissolved in distilled water) and an antibody diluent are mixed and used as the diluent for the reagents need to do the immunohistochemical staining.

 

Supplies:

 

Instant Nonfat Dry Milk
Available at any local grocery store


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Trypsin

Trypsin is a commonly used enzyme used for proteolytic-induced enzymatic retrieval (PIER) in immunohistochemistry. The trypsin reagent comes in a powder form and, therefore, should be dissolved in distilled water or an enzymatic diluent in order to be used on tissue samples. The end user should ultimately determine optimal working conditions (i.e., incubation times, temperatures, and concentrations) for the use of this product for immunohistochemical staining.

 

Supplies:

 

Trypsin from bovine pancreas
Sigma-Aldrich
www.sigmaaldrich.com
Catalog No. T9201


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Protease

Protease is an enzyme used for proteolytic-induced enzymatic retrieval (PIER) in immunohistochemistry. The protease reagent comes in a powder form and, therefore, should be dissolved in distilled water or an enzymatic diluent in order to be used on tissue samples. The end user should ultimately determine optimal working conditions (i.e., incubation times, temperatures, and concentrations) for the use of this product for immunohistochemical staining.

 

Supplies:

 

Protease from Streptomyces griseus
Sigma-Aldrich
www.sigmaaldrich.com
Catalog No. P5147


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Proteinase K

Proteinase K is an enzyme used for proteolytic-induced enzymatic retrieval (PIER) in immunohistochemistry. The IHC Select® Proteinase K is a reagent used for the enzymatic pretreatment of formalin-fixed, paraffin-embedded tissues. The datasheet for this product has protocol recommendations. However, the end user should ultimately determine optimal working conditions (i.e., incubation times, temperatures, and concentrations) for their immunohistochemical staining.

 

Supplies:

 

IHC Select® Proteinase K
Millipore
www.millipore.com
Catalog No. 21627


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Normal Rabbit IgG – Affinity Purified

A purified normal rabbit IgG should be used as the negative control reagent when performing immunohistochemical staining with a purified rabbit IgG primary antibody. The amount of IgG in this reagent should be matched to that of the primary antibody and then applied to the samples that will serve as negatives.

Suppliers:

Normal Rabbit IgG
EMD Millipore / Calbiochem
Billerica, Massachusetts 01821
www.millipore.com
1-800-645-5476
Catalog # NI01-100UG

IgG From Rabbit Serum
Sigma-Aldrich
St. Louis, MO
www.sigmaaldrich.com
1-800-325-3010
Catalog # I18140

Rapid Fixx

Rapid Fixx is a formalin-based product we use to fix slides with frozen sections.

This product is ready-to-use. Once the frozen tissue is sectioned and mounted on positively charged slides, the slides are immersed in the Rapid Fixx for seven seconds. They are immediately rinsed in tap water to remove excess fixative and then placed in buffer. (Note: The fixation time may be changed if you deem it necessary for your samples.)

Supplies:

Shandon Rapid Fixx Formalin
Thermo Fisher Scientific
www.fishersci.com
Catalog No. 67-642-12


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1X Trilogy™

Trilogy™ Pretreatment Solution is an antigen retrieval reagent that can be used on formalin-fixed, paraffin-embedded tissue sections. It comes as a 20X concentrate or in a ready-to-use form.

Prepare a working reagent from the concentrate by diluting it down to a 1X concentration using DI water.

Supplies:

Trilogy™ Pretreatment Solution
Cell Marque Corporation
www.cellmarque.com
Rocklin, CA 95677
800-665-7284
Catalog No. 920P


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1X Tris Buffer

Tris buffer is a pH 9.5 solution used in heat-induced epitope retrieval procedures.
Dilute the Nuclear Decloaker 10X down to a 1X concentration using deionized water.

Supplies:

Nuclear Decloaker, 10X
Biocare Medical
www.biocare.net
Concord, CA 94520
800-799-9499
Catalog No.CB911M

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