- Proteins and Peptides
- Lysates and Cell Lines
1. Cut 5 mm tissue sections and mount on a slide.
2. Deparaffinize formalin-fixed and paraffin-embedded tissue sections with three 10 min soaks in xylene, and then rehydrate with two 5 min soaks in 100% ethanol, two 5 minute soaks in 95% ethanol and two 5 minute soaks in 70% ethanol followed by a rinse (10 dips) in 1X PBS.
3. Block endogenous peroxidases with 0.3% H2O2 in PBS for 30 minutes followed by 3 rinses in 1X PBS.
Perform antigen retrieval:
1. Microwave in citrate buffer pH 5 for 5 minutes. Check buffer level and microwave again in citrate buffer pH 5 for another 5 minutes. Allow the slides to cool for 20 min and rinse 3X in 0.1% Trition-X in PBS.
2. Citrate buffer formula: 0.1M citric acid stock (21.01 g of citric acid in 1 L water) and 0.1M sodium citrate stock (29.41g of sodium citrate in 1 L water). Mix 9 mls of citric acid stock with 41 mls of sodium citrate stock and complete to 500 ml with distilled water.
(Note: citrate buffer is now available for purchase from several companies including BD Pharmingen and Biogenx).
Perform immunohistochemical staining according to the guidelines of the Catalyzed Signal Amplification System (DAKO, Carpinteria, CA, cat# K1500):
1. Block non-specific protein binding by setting slides in chamber with 1% BSA in PBS and incubating for 20 minutes.
2. For amplification, incubate with CSA kit Protein block (solution #2) for 5 minutes.
3. Incubate the slides with a 1:250 dilution of anti-hCTR1 antibody (NB 100-402), diluted in 1% BSA in PBS and incubate overnight at 4C.
4. Wash slides with 3X TBS/0.1% Tween.
5. Apply Rabbit Link antibody (from DAKO, Carpinteria, CA, Cat # K1498 ) for 1 hour, followed by 3X wash with TBS/0.1% Tween.
6. Apply prepared streptavidin complex (from DAKO CSA kit, solutions #5,6,7) for 15 minutes, followed by 3X wash with TBS/0.1% Tween.
(Note: the prepared streptavidin complex needs to be made fresh and needs to sit for 30 minutes prior to use).
7. Apply amplification reagent (from DAKO CSA kit, solution #8) for 15 minutes, followed by 3X wash with TBS/0.1% Tween.
8. Apply streptavidin peroxidase (from DAKO CSA kit, solution #9) for 15 minutes, followed by 3X wash with TBS/0.1% Tween.
9. Incubate with substrate: prepare fresh AEC (3-amino-9ethylcarbazol) (Vector Laboratories, Burlingame, California) and apply for 10-30 minutes followed by 3X wash with PBS.
10. Counterstain with Mayers Hematoxylin for 2 minutes followed by 3X wash with water.
11. Mount using Gelmount (aqueous, from Biomeda, cat #M0).
Several negative controls can be run in parallel with experimental samples. One control that has been used is to stain the tissue sections with non-immune rabbit IgG1 sera (DAKO, prediluted). As a technical positive control, slides can be incubated with anti-vimentin antibody (prediluted, DAKO, cat# V1613).
Further controls included staining of cells expressing higher levels of hCTR1, and using the immunizing peptide (1 mg/ml) at a 1:50 dilution to compete out the hCTR1 antibody, resulting in a negative stain.