To use the Visikol® HISTO-M™ approach with immunolabeling, several buffers are required which can either be purchased from Visikol through the Visikol store or can be made using common laboratory reagents.
Recipes for 100 mL – prepare in volumetric flask
| Buffer name | Composition | Recipe for 100 mL | Storage |
|---|---|---|---|
| PBST | · PBS (1X) · Tween® 20 (0.2% wt.)* | · 200 mg Tween® 20 · Dilute to 100 mL with 1X PBS | RT |
| Penetration Buffer | · PBS (1X) · 0.2% Tween® 20* · 0.3M Glycine · 20% DMSO | · 2.25 g Glycine · Add 20 mL 1X PBST · 20 mL DMSO · Dilute to 100mL with ~60 mL PBST | RT |
| 10X PTwH (10X washing buffer) | · 10X PBS · 2% Tween® 20 · 100 ug/mL Heparin | · 10 mg Heparin · Add 20 mL 10X PBS · 2g Tween 20 · Dilute to 100 mL with 10X PBS | 4°C, 6 months. Add 0.05% sodium azide for long term storage |
| Blocking buffer | · PBS · 0.2% Tween 20* · 6% Donkey Serum · 10% DMSO | · 6 mL Donkey Serum · Add 10 mL PBST · Add 10 mL DMSO · Dilute to 100 mL with PBST | 4° C Add 0.05% sodium azide for long term storage |
| Antibody buffer | · PBS · 0.2% Tween® 20* · Heparin · 3% Donkey Serum · 5% DMSO | · 3 mL Donkey Serum · Add 20 mL 1X Washing Buffer · Add 5 mL DMSO · Dilute to 100 mL with 1X Washing Buffer | 4° C Add 0.05% sodium azide for long term storage |
- To make 100 mL of 1X washing buffer, dilute 10 mL 10X buffer with 90 mL of DI H2O
- Sodium azide will interfere with horseradish peroxidase-based protocols, but has no effect on immunofluorescent labeling
Catalog numbers
- Tween® 20– Fisher Scientific – Catalog # BP337
- Glycine – Fisher Scientific – Catalog # G46
- DMSO – Fisher Scientific – Catalog # BP231
- Heparin – Alfa Aesar – Catalog # A16198
- Normal Donkey Serum – Jackson Immunoresearch – Catalog #: 017-000-121
*Tween is a registered trademark of Croda International PLC.