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Immunohistochemistry ESE3 Immunohistochemistry Slide 2 stained prostate tissue samples for ESE3 troubleshooted ESE3 antibody using the controls - no antibodies - primary antibody only - secondary antibody only - IgG and secondary antibody What I Did What is Immunohistochemistry? a method used to detect and achieve visualization of the presence and distribution of a specific antigen in cells or tissues uses antigen-antibody reactions, a ABC reagent and an enzyme substrate to achieve staining Slide 3 a nuclear protein a transcriptional factor epithelial-specific expression expressed in normal cells down-regulated in cancerous cells at both the mRNA and protein levels acts as a tumor suppressor due to its involvement in the control of cell differentiation ESE3 Slide 4 Immunohistochemistry Protocol Immunohistochemistry Protocol Slide 5 Deparaffinize and Rehydrate 1. 3 x10 min xylene; 2 x 2 min in 100% ETOH, 2 x 2 min in 70% ETOH. Make sure to shake off excess xylene on slide before putting the slides into EtOH in order not to dilute the EtOH. 2. Rinse sides gently with ddH 2 O squirt bottle being careful not to rinse directly on the section. Wash with ddH 2 O in Coplin jar for 2 x 2 min on shaker at 120 rpm. Quenching Endogenous Peroxidase 1. Wipe off excess ddH 2 O from slide - work quickly to ensure that section does not completely dry out. To do this shake the slide and use a kimwipe to get rid of any extra liquid. 2. Pipette enough hydrogen peroxide (3% H 2 O 2 in H 2 O) to cover the entire section and incubate for 10 min. Does not need to go into a humidity chamber. 3. Wash Sections with 1 x TBST with squirt bottle and then wash in Coplin jar for 2 x 2 min on shaker. Part 1 Slide 6 Antigen Retrieval 1. While section is incubating in hydrogen peroxide, pre-heat Antigen Retrieval Buffer in food steamer for 15 mins. Lift up the containers in order to fill the bottom of the steamer up with tap water, up to the second line. Put approximately 200 mL of antigen retrieval buffer into the glass slide holder in the steamer. 2. Incubate slides in pre-heated Antigen Retrieval Buffer for 20 min. Use forceps to transfer the slides into the slide holder. 3. Remove lid from the food steamer after the 20 min period and let the slides sit inside the steamer for 10 min. 4. Remove the entire slide box with buffer and let sit at room temperature on bench for 20 mins. 5. Wash slides in 1 x TBST for 3 x 2 min in Coplin jar on shaker Slide 7 Blocking and Primary Antibody Addition 1. Gently dry slides and put in humidity chamber which is tupperware lined with moist paper towels and a slide holder. Redraw hydrophobic barriers, if they come off. 2. Incubate slides for 1 hr at room temperature with blocking buffer in the humidity chamber: 10% rabbit serum (RS) and 1 x TBST. 5 slides 2x[1.00 mL of 1xTBST and 100 uL of RS] 3 or 4 slides 1.25 mL of 1xTBST and 125 uL of RS 3. Wipe and shake off excess blocking buffer. Add primary antibody diluted in blocking buffer and incubate overnight at 4 C in humidity chamber 5 slides (1:300) Rat IgG (1mg/mL) - 1:300 2x[3.33 uL of ESE3 in blocking buffer] 1.33 uL of Rat IgG in blocking buffer 3 or 4 slides (1:300) 4.16 uL of ESE3 in blocking buffer Slide 8 1. Wash slides 3 x 5 min in 1 x TBST 2. Add appropriate biotinylated secondary antibody to each section. Incubate in humidity chamber for 1 hr at room temperature. For anti- rat biotinylated antibody (1:200) 5 slides 5 uL in 1.0 mL of 1x TBST 3 or 4 slides 6.25 uL in 1.0 mL of TBST 3. During this incubation, prepare ABC reagent following manufacturers instructions or for a small volume add 12.5 uL of Reagent A to 1.25 mL of dilution buffer, TBST, then immediately add 12.5 uL of Reagent B and mix well. Allow for ABC reagent to sit at room temperature for about 30 min. 4. Wash Slides for 2 x 5 min in 1 x TBST on shaker. 5. Incubate sections with ABC reagent for 1 hr in humidity chamber. 6. Wash slides for 2 x 5 min in 1 x TBST on shaker. Part 2 - Visualization with Vector Labs ABC Kit Slide 9 7. Prepare DAB solution and add appropriate amount to slides. Incubate slides in DAB solution for 4 min In 5.0 mL of distilled H 2 O i. Add 2 drops of buffer, mix well ii. Add 4 drops of DAB stock solution, mix well iii. Add 2 drops of Hydrogen Peroxide, mix well 8. Wash slides under running distilled H 2 O for about 30 sec. Incubate in distilled H 2 O for 5 min. 9. Counterstain with appropriate amount of haematoxylin [HE] for 40 sec. Wash slides under running tap H 2 O for about 30 sec. Incubate in tap H 2 O for 5 min. 10. Clear and dehydrate slides: 2 x 3 min 70% EtOH, 2 x 3 min 100% EtOH, 2 x 3 min xylene. 11. Coverslip with Cytoseal mounting medium. Slide 10 Results Slide 11 40x 60x NormalCancerousPIN Slide 12 nuclear staining Normal Gland (40x) Slide 13 Cancerous Gland (40x) no nuclear staining Slide 14 PIN (40x) no nuclear staining nuclear staining Slide 15 Dr. Tang & Judy Yan supervisor Special Thanks To Slide 16 Slide 17 Slide 18 Slide 19 Slide 20