Immunofluorescent staining of formalin-fixed OCT-embedded human lung tissue
Jamie Verheyden, Justinn Barr, Xin Sun
Abstract
This protocol describes steps for immunofluorescent staining of formalin-fixed paraffin-embedded human lung tissue.
Steps
2X5 min wash in Phosphate-buffered saline with Tween (PBSTW) in a slide holder on a flat rotator.
Blocking:
Mix up 5% serum (50µL serum / 1mL of PBSTW)
Remove old PBSTW from the sample and add block.
Cover with Hybrislip.
Let sit in a humidified chamber at Room temperature for ≥ 1 hour.
Primary antibody:
Mix up more of the 5% serum.
Add antibody to the serum at the desired concentration.
Take the humidified chamber to a cold room.
Add primary antibody in the cold room.
Cover with Hybrislip.
Seal the humidified chamber.
On Day 2:
Wash slides 3X10 min at Room temperature in slide holder on a flat rotator.
Secondary antibody:
Mix up 5% serum.
Add secondary antibody to the serum at the desired concentration.
Add antibody mix to the slides.
Let it sit in a humidified chamber EITHER at Room temperature for 1 hour OR in the cold room overnight.
Cover with Hybrislip.
Wash:
1x10 min in PBSTW with DAPI.
2x10 min in PBSTW.
Mount the slides in VectaShield and seal with nail polish.
