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

Mercuric Chloride5 g
Potassium Dichromate2.5 g
ddH20100 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 Chloride5 g
Potassium Dichromate2.5 g
ddH20100 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.

Picric Acid (Saturated)75 ml
Formaldehyde (37-40%)25 ml
Glacial Acetic Acid5 ml

Carnoy’s Solution

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

Ethanol (100%)60 ml
Chloroform30 ml
Glacial Acetic Acid10 ml

Zinc Formalin

  1. Make a 0.1M Tris Buffer, pH 7.4
Trizma Tris Base12.1 g
1N HCL81.5 ml
ddH20900 ml

2. Prepare Zinc Fixative

Calcium Acetate0.5 g
Zinc Acetate5.0 g
Zinc Chloride5.0 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
Na2HPO421.8 g
NaH2PO46.4 g
  1. From this make a 0.1M Phosphate Buffer, pH 7.4
0.2M PB500 ml
  1. Proceed to make a 4% Paraformaldehyde in 0.1M Phosphate Buffer
Paraformaldehyde40 g
0.1M Phosphate Buffer1000 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

Formalin (37-40% stock solution)100ml
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
ddH201000 ml
Tween 200.5 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

EDTA0.37 g
ddH201 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)

Tris1.21 g
EDTA0.37 g
ddH201000 ml
Tween 200.5 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%)
Trypsin50 mg
ddH201000 ml

*Store at -20ºC

  1. Make 1% Calcium Chloride Stock Solution
Calcuim Chloride0.1 g
ddH201000 ml

*Store at 4ºC

  1. Trypsin working solution (0.05%)
Trypsin Stock Solution (0.05%)1 ml
Calcium Cholride stock Solution (1%)1 ml
Distilled Water8 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)

30% H2O210 ml
1X PBS90 ml

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

Peroxidase Blocking Soluiton (0.3% H2O2 in Methanol)

30% H2O2 1 ml
Methanol99 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

rabbit serum2%
cold fish skin gelatin0.1%
Triton X-1000.1%
Tween 200.05%
sodium azide0.05%
1M PBS, pH 7.2

*Store at 4ºC

Universal Blocking Buffer

cold fish skin gelatin0.1%
Triton X-1000.5%
sodium azide0.05%
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.