- Proteins and Peptides
- Lysates and Cell Lines
IHC Staining Protocol
1. Paraffin sections (4-6um) of animal or human tissue (material fixed with 10% phosphate buffered formalin)
2. Phosphate buffered saline (PBS) pH 7.5-7.6 (10-20 mM) containing 1% BSA
3. 0.1% Pronase in PBS
4. 0.1% Trypsin in PBS
5. 10 ug/ml Ulex Europaeus-1 lectin (NBP2-42700)
6. Anti Ulex Europaeus-1 lectin antibody (NB110-13922)
7. Secondary antiserum: FITC anti-rabbit IgG (H+L)
8. Mounting Medium
1. After deparaffinization treat with 0.1% Promase in PBS for 10 minutes at 37C. Alternatively, digest with 0.1% Trypsin in PBS for 25 minutes at 37C. Gently rinse in PBS for 5 minutes (2X).
2. Apply 2 drops of 10 ug/ml solution of UEA-1 (NBP2-42700)
3. Incubate for 30 minutes at 37C. Gently rinse in PBS for 5 minutes (X2)
4. Apply 2 drops Anti Ulex Europaeus-1 lectin antibody (NB110-13922) diluted 1:500 in PBS containing 1% BSA
5. Incubate for 1 hr. at 37C
6. Gently rinse in PBS for 5 minutes
7. Drain, carefully blot away excess moisture around the sections.
8. Quickly apply freshly prepared diluted FITC secondary antiserum (at previously determined appropriate dilution) in PBS containing 1% BSA.
9. Incubate for 30 minutes at 37C
10. Gently rinse in PBS for 5 minutes (x2). Blot away excess moisture from around the sections.
11. Add mounting medium and coverslip.
12. Read under the UV fluorescent microscope. Mounted preparations can be stored in a refrigerator.
1. Do not allow tissue sections to dry out at any time during the aforementioned procedure.
2. In case of excessive background staining remove aggregates from antisera by centrifuging for 15 minutes immediately prior to use.