Immunofluorescent Labeling

Immunofluorescent labeling for fresh-frozen, cryosectioned tissue: Adapted by KPA from MK IF protocol for floating sections

Use this worksheet.

Set-Up:

1. EasyDip™ Slide Staining System (EMS) for formaldehyde fixation

2. Prepare 4% formaldehyde solution in PBS (see white binder in processing room)
3. 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:

1. Quickly load cold slides into slide rack (grey), then place slide rack in holder (white, "trash can") containing 4% PFA for 20 minutes [  ]
2. Once fixed, remove slide rack and dunk in new holder containing PBS
3. Removed slides from rack and place on slide staining tray (black with aluminum lid, like this) 
4. 1% H₂O₂ in PBS, 20 min [   ]
5. PBS washes, until bubbles disappear on rotator at slow speed [   ]
6. Blocking: 10% NGS + 1% BSA + 0.3% Triton X-100 in PBS, 1 hr [   ]

1. 1° Ab in PBS containing 1% NGS, 1% BSA, 0.3% Triton X-100, overnight at RT [   ]

Start time =                           à End time =

1. Antibody 1 =              anti-                       , vendor, Cat#, lot#:
   • Dilution = 1:          =             µl/well
2. Antibody 2 =           anti-                       , vendor, Cat#, lot#:      
   • Dilution = 1:          =             µl/well

Day 2:

1. PBS wash, 10 min × 3 [   ]  [   ]  [   ]
2. 2° Ab in PBS with 1% BSA, 1% NGS, 0.3% Triton X-100, 1 hr at RT [   ]
   1. Antibody 1 = anti-                   , vendor, Cat#, lot#:
      • Dilution = 1: =             µl/well
   2. Antibody 2 = anti-                   , vendor, Cat#, lot#:      
      • Dilution = 1: =             µl/well

1. PBS wash, 10 min × 3 [   ]  [   ]  [   ]
2. DAPI, 5 min [   ]
   • stock solution = 1 mg/ml in purified water
   • working solution = dilute stock solution 1:1,000 in PBS
3. PBS wash, 10 min × 3 [   ]  [   ]  [   ]
4. Aqua/Poly anti-fade mountant and cover slip