IHC Buffers & Recipes
Immunohistochemistry (IHC) 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
- 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
- Make a 0.2M Phosphate Buffer (PBS) , pH 7.4
| Reagent | Amount |
|
NaHPO
|
21.8 g
|
|
NaHPO
|
6.4 g
|
|
ddH20 |
1000ml
|
2. From this make a 0.1M Phosphate Buffer, pH 7.4
| Reagent | Amount |
|
0.2M PB
|
500 ml
|
|
ddH2-
|
500ml
|
3. 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
- Make a trypsin stock solution (0.05%)
| Reagent | Amount |
|
Trypsin
|
50 mg
|
|
ddH20 |
1000 ml
|
*Store at -20ºC
- Make 1% Calcium Chloride Stock Solution
| Reagent | Amount |
|
Calcuim Chloride
|
0.1 g
|
|
ddH20
|
1000 ml
|
*Store at 4ºC
- 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 for 10 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.