Immunohistochemistry Buffers & Recipes

Outlined below you will find a selection of immunohistochemistry buffers and recipes including: fixation buffers, buffers for heat-induced epitope retrieval (HIER), enzymatic detection buffers, permeabilization and blocking buffers.

Sample Fixation Buffers

Zenker’s Solution

Use Zenker’s solution for bloody samples such as spleen and connective tissue

Reagent Amount
Mercuric Chloride 5 g
Potassium Dichromate 2.5 g
ddH20 100 ml

*Heat and cool. Add 5 ml of glacial acetic acid just before use. Never use metal forceps when handling tissues fixed in Zenker Solution.

Helly’s Solution

Helly’s solution can be used on blood forming organs such as bone marrow, liver and spleen.

Reagent Amount
Mercuric Chloride 5 g
Potassium Dichromate 2.5 g
ddH20 100 ml

*Heat and cool. Add 5 ml of formaldehyde just before use.

Bouin’s Fixative

Bouin’s Fixative should be used when soft tissue structures need to be preserved.

Reagent Amount
Picric Acid (Saturated) 75 ml
Formaldehyde (37-40%) 25 ml
Glacial Acetic Acid 5 ml

Carnoy’s Solution

Carnoy’s solution should be used for the fixation of DNA and RNA.

Reagent Amount
Ethanol (100%) 60 ml
Chloroform 30 ml
Glacial Acetic Acid 10 ml

Zinc Formalin

  1. Make a 0.1M Tris Buffer, pH 7.4
Reagent Amount
Trizma Tris Base 12.1 g
1N HCL 81.5 ml
ddH20 900 ml

2. Prepare Zinc Fixative

Reagent Amount
Calcium Acetate 0.5 g
Zinc Acetate 5.0 g
Zinc Chloride 5.0 g
0.1M Tris Buffer (see above) 1000 ml

* Mix to dissolve. Adjust the final pH to 6.5-7.0. Store the at room temperature

4% Formaldehyde Fixative Solution

  1. Make a 0.2M Phosphate Buffer (PBS) , pH 7.4
Reagent Amount
Na2HPO4 21.8 g
NaH2PO4 6.4 g
ddH20 1000ml
  1. From this make a 0.1M Phosphate Buffer, pH 7.4
Reagent Amount
0.2M PB 500 ml
ddH2- 500ml
  1. Proceed to make a 4% Paraformaldehyde in 0.1M Phosphate Buffer
Reagent Amount
Paraformaldehyde 40 g
0.1M Phosphate Buffer 1000 ml

* Heat to 60-65 ºC while stirring. Add a few drops of 1N NaOH until solution clear. Continue stirring until the solution is dissolved. Cool and filter.

10% Neutral Buffered Formalin

Reagent Amount
Formalin (37-40% stock solution) 100ml
ddH20 900ml
NaH2PO4 (monobasic) 4g/L
Na2HPO4 (dibasic/anhydrous) 6.5g/L

* 10% formalin represents 10% of the 37-40% stock solution. The actual amount of dissolved formaldehyde in the 10% formalin is therefore only 3.7-4.0%.

Buffers for heat-induced epitope retrieval (HIER)

Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0)

Reagent Amount
Tri-sodium citrate (dihydrate) 2.94 g
ddH20 1000 ml
Tween 20 0.5 ml

*Adjust pH to 6.0 with 1N HCL. Store at room temperature for 3 months, for longer storage store at 4ºC

1 mM EDTA, pH 8.0

Reagent Amount
EDTA 0.37 g
ddH20 1 L

*Adjust pH to 8.0 with NaOH, store at room temperature for 3 months.

Tris-EDTA buffer (10mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0)

Reagent Amount
Tris 1.21 g
EDTA 0.37 g
ddH20 1000 ml
Tween 20 0.5 ml

*Adjust pH to 9.0. Store at room temperature for 3 months, for longer store at 4ºC

Buffers for enzymatic antigen retrieval

Trypsin Buffer

  1. Make a trypsin stock solution (0.05%)
Reagent Amount
Trypsin 50 mg
ddH20 1000 ml

*Store at -20ºC

  1. Make 1% Calcium Chloride Stock Solution
Reagent Amount
Calcuim Chloride 0.1 g
ddH20 1000 ml

*Store at 4ºC

  1. Trypsin working solution (0.05%)
Reagent Amount
Trypsin Stock Solution (0.05%) 1 ml
Calcium Cholride stock Solution (1%) 1 ml
Distilled Water 8 ml

*Adjust the pH to 7.8 with NaOH, store at 4 ºC for one month for longer store at -20 ºC.

Permeabilization Buffers

Triton or NP-40

Use 0.1 – 0.2 % in PBS for10 minutes only

*Partially dissolves the nuclear membrane for nuclear antigen staining.

Tween 20, Saponin, Digitonin and Leucoperm

Use 0.2 – 0.5% for 10-30 minutes.

*Suitable for cytoplasmic antigens and soluble nuclear antigens.

Blocking Buffers

Peroxidase Blocking Solution (3% H202 in PBS)

Reagent Amount
30% H2O2 10 ml
1X PBS 90 ml

*Store at 4ºC for up to 3 months. Recommended solution for paraffin sections.

Peroxidase Blocking Soluiton (0.3% H2O2 in Methanol)

Reagent Amount
30% H2O2  1 ml
Methanol 99 ml

*Store at 4 ºC. Recommended for Frozen sections.

Biotin blocking buffer

Biotin 0.001% in PBS.

*Store at 4 ºC

Blocking Sera Sample Buffer Recipes

Normal Rabbit Sera blocking buffer

Reagent Amount
rabbit serum 2%
BSA 1%
cold fish skin gelatin 0.1%
Triton X-100 0.1%
Tween 20 0.05%
sodium azide 0.05%
1M PBS, pH 7.2

*Store at 4ºC

Universal Blocking Buffer

Reagent Amount
BSA 1%
cold fish skin gelatin 0.1%
Triton X-100 0.5%
sodium azide 0.05%
0.01M PBS, pH 7.2-7.4

*Store at 4 ºC. Do not use to dilute HRP conjugated antibodies as sodium azide is an inhibitor of HRP.