Immunofluorescence (Immunocytochemistry), Blocking Buffer-free Protocol with Formaldehyde Fixation
IMPORTANT: This protocol is designed for unconjugated primary antibodies that are sensitive to serum blocking. When applicable, it will be linked under the Product Information section on an antibody's product-specific webpage.
A. 溶液与试剂
NOTE: 用反渗透去离子 (RODI) 水或等效净化水制备溶液。
- 1X Phosphate Buffered Saline (PBS) : 配制 1 L 的 1X PBS：add 100 ml 10X PBS ( #12528 ) to 900 ml water, mix. 调节 pH 至 8.0。
- 4% Formaldehyde, Methanol-Free ( #47746 ): 使用新鲜溶液。Dilute concentrated stocks in 1X PBS.
- Antibody Dilution Buffer : Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer ( #12378 ), or prepare a 1X PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 30 µl Triton™ X-100 and 0.1 g BSA to 10 ml 1X PBS. 混匀。
- Fluorochrome-conjugated Secondary Antibody: 使用对您的一抗宿主物种（例如兔子）具有反应性的二抗。 Click here for a list of secondary antibodies approved for immunofluorescence.
- Mounting medium: Prolong ® Gold AntiFade Reagent ( #9071 ) or Prolong ® Gold AntiFade Reagent with DAPI ( #8961 ).
B. 固定
- Fix cells with 4% formaldehyde for 15 minutes at room temperature.
- Aspirate fixative, wash three times in 1X PBS for 5 minutes each.
- Proceed with Section C.
C. 免疫染色
NOTE: Do not allow cells to dry at any time during this procedure and protect sensitive fluorophores from light.
- Aspirate 1X PBS and then permeabilize cells with Antibody Dilution Buffer for 60 minutes.
- Prepare primary antibody in Antibody Dilution Buffer (see product website for recommended dilution range).
- Aspirate Antibody Dilution Buffer, apply diluted primary antibody.
- 4°C 孵育过夜。
- Wash three times with 1X PBS for 5 minutes each.
- Incubate cells in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in the dark.
- Wash three times in 1X PBS for 5 minutes each.
- Counterstain as appropriate and then mount samples for imaging.
- For long term storage, store stained samples at 4°C protected from light.