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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% PFA 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 -80C 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 [   ]

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