Immunofluorescence Method
Frozen Sections
Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 º C.
Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 º C until needed.
Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
Wash in PBS
Paraffin Sections
Procedure for Immunofluorescence Staining
1. Rinse Sections in washing buffer for 2x2 min.
2. Primary Antibody (没有标记一抗): incubate sections in primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight. Note
o not rinse sections between serum block and primary antibody incubation.
3. Rinse in washing buffer for 3x2 min.
4 Secondary Antibody: incubate sections in biotinylated secondary antibody (生物素标记的二抗) (1:500, Vector Labs) in secondary antibody dilution buffer for 30 minutes at room temperature.
5. Rinse in washing buffer for 3x2 min.
6. Detection: incubate sections in FITC-Avidin D (1:500, Vector Labs) in PBS for 30 minutes at room temperature. Protecting slides from light starting from this step to the end by covering slides with aluminum foil or black box.
7. Rinse in washing buffer for 3x2 min.
8. Counterstain with PI or DAPI if desired for 20-30 minutes at room temperature.
9. Rinse in washing buffer for 3x2 min.
10. Coverslip with Vector aqueous Anti-fade fluorescent mounting medium and seal with nail polish.
11. Store slides in dark at 4 ºC.
