7TM Immunohistochemistry Protocol
Note: This protocol is designed for the staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections.
1. Buffers and Reagents
Use double distilled water for buffer preparation or water with the same grade of purity.
- Citrate buffer: C6H8O7 x H2O: 1.8 mM, C6H5Na3O7 x H2O: 8.7 mM, pH 6.0
- PBS: Phosphate Buffered Saline (NaCl: 150 mM, KH2PO4: 50 mM)
- PBS/BSA: PBS with 1% BSA (bovine serum albumin)
2. Deparaffinization, Rehydration and Antigen Unmasking
- Wash sections with xylene for 20 min. Aspirate xylene. Repeat 3-times.
- Wash sections with 100% ethanol for 20 min. Aspirate ethanol. Repeat 3-times.
- Blocking of endogenous peroxidase: Incubation with 0.15% H2O2 in methanol for 45 min.
- Wash sections with 95% ethanol for 2 min. Aspirate ethanol. Repeat 2-times.
- Wash sections with 80% ethanol for 2 min. Aspirate ethanol.
- Wash sections with 70% ethanol for 2 min. Aspirate ethanol.
- Wash sections with water for 5 min. Aspirate water. Repeat 2-times.
- Use a microwave for the following boiling/cooling cycles in citrate buffer:
- boil for 8 min
- cool at room temperature for 4 min
- boil for 4 min
- cool at room temperature for 4 min
- boil for 4 min
- cool at room temperature for 20 min
- aspirate citrate buffer and incubate for 5 min in PBS/BSA.
3. Staining and Mounting
- We recommend the use of a Shandon™ Sequenza™ staining rack for the following steps.
- Wash with 1.5 ml PBS/BSA for 5 min.
- Incubate sections with 300 µl 7TM Premium IHC-Grade Antibodies at a dilution of 1:100 in PBS/BSA for 1-2 hours at room temperature or at 4 °C overnight (we recommend the incubation overnight).
- Wash with 1.5 ml PBS/BSA for 5 min.
- Incubate sections with 150 µl biotinylated anti-rabbit-IgG of your choice for 20 min.
- Wash with 1.5 ml PBS/BSA for 5 min.
- Incubate sections with 150 µl peroxidase-conjugated streptavidin of your choice for 20 min.
- Wash with 1.5 ml PBS/BSA for 5 min.
- Incubate in 3-amino-9-ethylcarbazole (AEC) solution of your choice for 15 min. Repeat 2-times.
- Wash with 1.5 ml water for 5 min.
- Wash with water for 1 min. Repeat 2-times.
- Incubate sections with hematoxylin solution of your choice for 5 min.
- Wash with water for 2.5 min
- Dip sections in 250 ml water containing 1.4 ml concentrated ammonia. Repeat 5-times.
- Wash with water for 2.5 min.
- Mount sections by using an aqueous mounting media on microscope slides.
For more information please contact us:
E-Mail: support@7tmantibodies.com
Fon: 0049-151-20130575
FAX: 0049-3641-2414958