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Immunohistochemistry protocol validated for the antibodies described below:
ADI-905-781, ADI-KAP-GP005, ADI-VAM-PT046, ADI-VAM-SV016, ADI-VAM-SV020, ADI-VAM-SV021, ADI-VAM-SV026, ADI-VAP-PT049, ADI-VAP-SV065, ADI-AAP-113, ADI-CSA-815, ADI-OSA-110, ADI-SOD-110, ADI-SPA-200, ADI-SPA-222, ADI-SPA-601, ADI-SPA-800, ADI-SPA-803, ADI-SPA-806, ADI-SPA-815, ADI-SPA-816, ADI-SPA-827, ADI-SPA-830, ADI-SPA-835, ADI-SPA-846, ADI-SPA-850, ADI-SPA-860, ADI-SPA-890, ADI-SPA-894, ADI-SPA-896, ADI-AAP-240, ADI-KAP-MA001, ADI-KAP-PK004, ADI-KAP-PK005, ADI-KAP-PK112, ADI-KAP-ST002, ADI-OSA-150, ADI-SOD-100, ADI-SOD-111, ADI-SPA-203, ADI-SPA-223, ADI-SPA-400, ADI-SPA-550, ADI-SPA-585, ADI-SPA-757, ADI-SPA-820, ADI-SPA-829, ADI-SPA-836, ADI-SPA-851, ADI-SPA-865, ADI-SPA-895, ADI-SPA-950, ADI-905-165, ADI-905-678, ADI-905-682, ADI-905-684, ADI-AAM-127, ADI-AAM-137, ADI-AAM-149, ADI-AAM-175, ADI-CSA-620, ADI-KAP-CA002, ADI-KAP-CC203, ADI-KAP-CP130, ADI-KAP-NO020, ADI-KAP-NO030, ADI-KAS-MA013, ADI-KAS-ST009, ADI-SPA-110, ADI-SPS-720
Tissue Preparation:
Formalin fixation and embedding in paraffin wax
Tissue Sectioning:
Make 4-µm sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C.
Deparaffinization:
Wash dry slides in three changes of xylene – 5 minutes each at room temperature
Rehydration:
Wash slides in three changes of 100% alcohol – 3 minutes each at room temperature
Wash slides in two changes of 95% alcohol – 3 minutes each at room temperature
Wash slides in one change of 80% alcohol – 3 minutes at room temperature
Rinse slides in gentle running distilled water – 5 minutes at room temperature
Antigen retrieval:
Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C – 20 minutes
Remove from heat and let stand at room temperature in buffer – 20 minutes
Rinse in 1X TBS with Tween (TBST) – 1 minute at room temperature
Immunostaining:
(Do not allow tissues to dry at any time during the staining procedure)
Apply a universal protein block – 20 minutes at room temperature
Drain protein block from slides, apply diluted primary antibody – 45 minutes at room temperature
Rinse slides in 1X TBST – 1 minute at room temperature
Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes at room temperature
Rinse slides in 1X TBST – 1 minute at room temperature
Apply alkaline phosphatase streptavidin – 30 minutes at room temperature
Rinse slides in 1X TBST – 1 minute at room temperature
Apply alkaline phosphatase chromogen substrate – 30 minutes at room temperature
Wash slides in distilled water – 1 minute at room temperature
Dehydrate:
(This method should only be used if the chromogen substrate is alcohol insoluble (e.g. HIGHDEF® DAB Chromogen/Substrate Set)
Wash slides in two changes of 80% alcohol – 1 minute each at room temperature
Wash slides in two changes of 95% alcohol – 1 minute each at room temperature
Wash slides in three changes of 100% alcohol – 1 minute each at room temperature
Wash slides in three changes of xylene – 1 minute each at room temperature
Apply coverslip