免疫荧光处理流程

NOTE: Prepare solutions with purified water.

1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride

(NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 8.0. 2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh, store

opened vials at 4°C in dark, dilute in PBS for use.

3. Blocking Buffer: (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To

prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal serum from the same species as the secondary antibody (e.g., normal goat serum, normal donkey serum) and 21.25 ml dH2O and mix well. While stirring, add 75 µl Triton™ X-100.

4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 40

ml, add 4 ml 10X PBS and 120 µl Triton™ X-100 to 0.4 g BSA. Bring to final volume of 40 ml with dH2O and mix well.

5. Fluorochrome-conjugated secondary antibodyNOTE: When using any primary or

fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

6. Prolong® Gold Anti-Fade Reagent (#9071), with DAPI (#8961).

. Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in PBS.

NOTE: Formaldehyde is toxic, use only in fume hood. 2. Allow cells to fix for 15 min at room temperature.

3. Aspirate fixative, rinse three times in PBS for 5 min each. 4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

1. Block specimen in Blocking Buffer for 60 min.

2. While blocking, prepare primary antibody by diluting as indicated on datasheet in

Antibody Dilution Buffer.

3. Aspirate blocking solution, apply diluted primary antibody. 4. Incubate overnight at 4°C.

5. Rinse three times in PBS for 5 min each.

NOTE: If using primary antibodies directly conjugated with Alexa Fluor®fluorochromes, then skip to (Section C, Step 8).

6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody

Dilution Buffer for 1–2 hr at room temperature in dark. 7. Rinse in PBS (Section C, Step 5).

8. Coverslip slides with Prolong® Gold Anti-Fade Reagent (#9071), with DAPI (#8961). 9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

紫外光(UV)：激发光谱区域：330-400nm 紫光(V)：激发光谱区域：395-415nm 蓝光(B)：激发光谱区域：420-485nm 绿光(G)：激发光谱区域：460-550nm

Leica DM3000 荧光显微镜，光源是30W透射光和100W落射汞灯。若是Leica DM1000 LED光源是LED透射光和50W落射汞灯 配置里的三个荧光虑片为：

（1）filter system A S 紫外激发（激发：BP 340/380；发射：LP 425） （2）filter system I3 S 蓝光激发 (激发：BP 450/490；发射：LP 515) （3）filter system N2.1 S 绿光激发 (激发：515-560；发射：LP 590)