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Immunohistochemistry

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


Much of the work carried out by DTT is in support of the National Toxicology Program (NTP), an interagency partnership of the Food and Drug Administration, National Institute for Occupational Safety and Health, and NIEHS.

Visit the NTP Website

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

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)

Magnesium Chloride Solution

  • Sigma-Aldrich
    St. Louis, MO 63178
    800-325-3010
    Catalog No. M1028

Manganese (II) Chloride Solution

  • Sigma-Aldrich
    St. Louis, MO 63178
    800-325-3010
    Catalog No. M1787

Tween 20

  • Bio-Rad
    Hercules, CA 94547
    800-424-6723
    Catalog No. 170-6531

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
    888-584-8929
    Catalog No. P36934

ProLong® Gold Antifade Reagent With DAPI Life Technologies

  • Invitrogen
    Grand Island, NY 14072
    888-584-8929
    Catalog No. P36935

1X Wash Buffer

Biocare Medical TBS Automation Wash Buffer is supplied as a 500 mL concentrated Tris-buffered saline solution (20X) containing Tween-20, pH 7.7 (±0.1). It is sufficient for preparing 10 L of working solution.

Prepare 1X Wash Buffer by mixing 500 mL of 20X concentrate with 9.5 L of deionized water.

Supplies:

TBS Automation Wash Buffer, 20X

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

1% BSA Diluent

BSA diluent (1%) is used in preparing many of the reagents for immunohistochemical 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)

TBS Automation Wash Buffer, 20X

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

  • Agilent Dako
    Carpinteria, CA 93013
    800-235-5763
    Code K3468

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

Rodent Decloaker, 10X

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)

Sodium Borate (Na2B4O7.10H2O)

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

Calcium Chloride Dihydrate (CaCl2⋅2H2O)

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

ChromPure Goat IgG, whole molecule

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%)

PLP Fixative

Part A - Making stock solutions. (All solution. 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 solution.
    6. Slowly add remaining 40mL of water.
    7. Filter solution. 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 solution (#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)

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.

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.

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

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

Hydrochloric Acid (HCl) (37%)

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

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

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

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

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

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

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

  • MilliporeSigma
    Burlington, MA 01803
    800-645-5476
    Catalog # NI01-100UG

IgG From Rabbit Serum

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

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

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