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        IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS

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        867
        DAY ONE
        Paraffin sections:
        1) Deparaffinize: Xylene , 3x, 5 min each
        2) Rehydrate: 100% EtOH, 2x, 5 min each
        95% EtOH, 2x, 5 min each
        80% EtOH, 1x, 5 min
        50% EtOH, 1x, 5 min
        Proceed to Antigen Retrieval below (step 3)
        Cryo sections (starting with slides that have been baked @ 50°C, 30min):
        1) Fix in 4% PFA/PBS, 15min
        2) rinse with PBS, 3x, 5 min each
        Proceed to Antigen Retrieval below (step 3)
        3) Antigen Retrieval: [Vector Labs: Antigen Unmasking Solution #H3300]
        For 200 mL: 200mL dH2O
        2 mL unmasking solution
        a) Fill a rectangular dish 1/3 full with dH20. This will be used as a water
        bath for the glass jar containing the diluted unmasking solution. Place
        glass jar containing above solution in the bath and heat in microwave
        until unmasking solution is visibly boiling � about 5-8 min. (I usually
        start preparing this when I’m towards the end of the rehydration steps
        outlined above)
        b) Place slides from step 2 into hot unmasking solution. Cover with lid.
        Transfer entire jar from water bath to bench top. Leave covered for 5 min
        at RT.
        c) Cool at RT, 5 min with glass lid off.
        d) rinse in dH2O, 2x, 5 min each
        4) Quench: 13.3 mL 30% H2O2
        186.7 mL PBS
        200 mL PBS/ 2% H2O2
        a) quench 1 min
        b) rinse in PBS, 5 min
        IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS
        USING VECTASTAIN ABC ELITE KIT:
        5) Block:
        a) Block I: 100 mL PBS
        1 g Bovine Albumin (Sigma A-9418)
        PBS + 1% BSA = PBA
        1 hr, RT
        b) Block II: PBA
        2% serum from animal used to produce 2° antibody (provided
        by Vectastain Kit, Vector Labs)
        150 μl per slide
        30 min, RT
        6) 1° Antibody:
        a) dilution of choice in PBA
        b) humidity chamber, 4°C, overnight
        DAY TWO
        1) rinse slides in PBS, 2x, 5 min each
        2) 2° Antibody: [biotinylated, VectaStain kit]
        a) 1:200 dilution in PBA (ex. 5μl antibody per 1000μl PBA)
        b) humidity chamber, RT, 1 hr
        ~undefinedMEANWHILundefined make avidin-biotinylated peroxidase conjugate:
        5 mL PBS
        2 drops Reagent A. mix well
        2 drops Reagent B. mix well
        VectaStain Elite ABC Reagent
        Let sit at RT, 30 min before use
        c) rinse slides in PBS, 2x, 5 min each
        3) VectaStain Conjugation:
        a) 30 min, RT
        IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS
        USING VECTASTAIN ABC ELITE KIT:
        b) rinse in PBS, 2x, 5 min each
        4) Visualization: DAB Substrate Kit for Peroxidase [VectorLabs #SK4100]:
        5 mL dH2O
        2 drops DAB substrate buffer. Mix well
        4 drops DAB. Mix well
        2 drops H2O2. Mix well
        2 drops nickel-cobalt solution. Mix well
        a) use immediately upon making solution, 2-10 min usually enough to stain
        b) stop reaction by rinsing with dH2O, 5 min
        c) counterstain if desired
        5) Dehydrate: 95% EtOH, 2x, 2 min each
        100% EtOH, 2x, 2 min each
        Toluene, 2x, 2 min each
        6) Mount in Permount. Dry overnight
        <center> <p>  </p> </center>
        上一篇:Immunohistochemistry Protocol-Cytokine Antibodies   下一篇:免疫组化技术-Immunohistochemical Protocols
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