Immunofluorescent labeling for fresh-frozen, cryosectioned tissue: Adapted by KPA from MK IF protocol for floating sections
Use this worksheet.
Set-Up:
EasyDip™ Slide Staining System (EMS) for formaldehyde fixation
- Prepare 4% formaldehyde solution in PBS (see white binder in processing room)
- Using a plethora of dry ice to keep slides cold, locate and confirm existence of all slides to be used in the experiment from -80°C freezer
Day 1:
- Quickly load cold slides into slide rack (grey), then place slide rack in holder (white, "trash can") containing 4% PFA for 20 minutes [ ]
- Once fixed, remove slide rack and dunk in new holder containing PBS
- Removed slides from rack and place on slide staining tray (black with aluminum lid, like this)
- 1% H2O2 in PBS, 20 min [ ]
- PBS washes, until bubbles disappear on rotator at slow speed [ ]
- Blocking: 10% NGS + 1% BSA + 0.3% Triton X-100 in PBS, 1 hr [ ]
...
Per slide
...
For total of slides
...
BSA
...
15 mg
...
mg
...
NGS
...
15 µl
...
µl
...
Triton X-100 (3% stock)
...
150 µl
...
µl
...
PBS
...
1200 µl
...
µl
...
Total
...
1500 µl
...
µl
- 1° Ab in PBS containing 1% NGS, 1% BSA, 0.3% Triton X-100, overnight at RT [ ]
Start time = à End time =
- Antibody 1 = anti- , vendor, Cat#, lot#:
- Dilution = 1: = µl/well
- Antibody 2 = anti- , vendor, Cat#, lot#:
- Dilution = 1: = µl/well
...
Per slides
...
For total of slides
...
BSA
...
15 mg
...
mg
...
NGS
...
15 µl
...
µl
...
Triton X-100 (3% stock)
...
150 µl
...
µl
...
PBS
...
1200 µl
...
µl
...
Total
...
1515 µl
...
µl
Day 2:
- PBS wash, 10 min × 3 [ ] [ ] [ ]
- 2° Ab in PBS with 1% BSA, 1% NGS, 0.3% Triton X-100, 1 hr at RT [ ]
- Antibody 1 = anti- , vendor, Cat#, lot#:
- Dilution = 1: = µl/well
- Antibody 2 = anti- , vendor, Cat#, lot#:
- Dilution = 1: = µl/well
- Antibody 1 = anti- , vendor, Cat#, lot#:
...
Per slide
...
For total of slides
...
BSA
...
15 mg
...
mg
...
NGS
...
15 µl
...
µl
...
Triton X-100 (3% stock)
...
150 µl
...
µl
...
PBS
...
1200 µl
...
µl
...
Total
...
1515 µl
...
µl
- PBS wash, 10 min × 3 [ ] [ ] [ ]
- DAPI, 5 min [ ]
- stock solution = 1 mg/ml in purified water
- working solution = dilute stock solution 1:1,000 in PBS
- PBS wash, 10 min × 3 [ ] [ ] [ ]
- Aqua/Poly anti-fade mountant and cover slip