{"content":[{"text":"PROTOCOL OF PARAFFIN SECTIONS FOR IMMUNOSTAINING \n by JYP 8/13/02","bbox":{"left":0.172843,"top":0.091463,"width":0.654279,"height":0.03644499999999999}},{"text":"Deparaffinization","bbox":{"left":0.117647,"top":0.145829,"width":0.15911800000000004,"height":0.016926999999999998}},{"text":"Use glass sink \n 1. Dunk the section in Xylene, 5 minutes X 3 times. \n 2. Dunk in 100% ETOH, 5 minutes X 2 times. \n 3. Dunk in 90% ETOH, 5 minutes X 2 times. \n 4. Dunk in ddH2O X 1. \n 5. Boil the section in citrate buffer @ 95C X 10 minutes. \n 6. Cool down @ RT X 30 minutes.","bbox":{"left":0.117647,"top":0.180602,"width":0.501506,"height":0.122609}},{"text":"Fixation","bbox":{"left":0.117647,"top":0.321208,"width":0.075215,"height":0.016927000000000025}},{"text":"1. \n 2. \n 3. \n 4. \n 5. \n 6.","bbox":{"left":0.117647,"top":0.355905,"width":0.016326000000000007,"height":0.10404799999999997}},{"text":"Dunk the section in cold acetone X 5 minutes. \n Dunk the section in acetone/chloroform (1:1) X 5 minutes. \n Dunk the section in acetone X 5 minutes. \n Wash with PBS, 2 minutes X 2. \n Circle the section with Pap-pen. \n Place slide in a humidity chamber (by placing wet paper under the slide) with \n section covered by PBS.","bbox":{"left":0.147059,"top":0.355905,"width":0.6687970000000001,"height":0.12147200000000002}},{"text":"Primary and Secondary antibody incubation","bbox":{"left":0.117647,"top":0.495375,"width":0.41079899999999997,"height":0.016927000000000025}},{"text":"1. Cover the section with the primary antibody (diluted in goat serum: 1:250) X 1-3hr. \n @ RT or O.N. \n 2. Wash with PBS, 2 minutes X 3. \n 3. Incubate the section with goat serum X 10 minutes. \n 4. Cover the section with secondary antibody (diluted in goat serum: 1:1000) X 1-3hr. \n @ RT. \n 5. Wash with PBS, 2 minutes X 3.","bbox":{"left":0.117647,"top":0.530147,"width":0.748632,"height":0.12586699999999995}},{"text":"DAB staining","bbox":{"left":0.117647,"top":0.674011,"width":0.123052,"height":0.016927000000000025}},{"text":"1. \n 2. \n 3. \n 4. \n 5.","bbox":{"left":0.117647,"top":0.708784,"width":0.016326000000000007,"height":0.08662400000000003}},{"text":"Cover the section with SA-HRP (diluted in goat serum: 1:500) X 30 minutes. \n Wash X 2 with PBS. \n Cover the section with DAB mixture٭; monitor the color development (dark brown). \n Wash with PBS X 3minutes X 2. \n Counterstain with Light Green working solution, monitor the color change (best \n result should have both light green and dark brown). \n Dehydrate by alcohol: Cover the section with 95% ETOH X 5 minutes, remove; \n Cover the section with 100% ETOH X 5 minutes, remove; then Xylene X 5 minutes \n X 2. \n Mount the slide with permount, cover the section with cover slip.","bbox":{"left":0.147059,"top":0.708784,"width":0.7184159999999999,"height":0.17366900000000007}},{"text":"6.","bbox":{"left":0.117647,"top":0.813253,"width":0.016326000000000007,"height":0.016927000000000025}},{"text":"7.","bbox":{"left":0.117647,"top":0.865526,"width":0.016326000000000007,"height":0.016927000000000025}}],"images_bbox":[],"images_bbox_not_text_overlap":[]}