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Materials:

fixative	dependent on antigen (common: 4% formaldehyde or 100% MeOH)
post-fixative	dependent on antigen (usually MeOH)
PBS	1X PBS
PBST	PBS + detergent (0.05 to 0.5 % Tween or Triton)
DAPI (1000X)	1 ug/ml in EtOH
Antifade (BIORAD)
Block	1X PBS 1% BSA 10% Donkey Serum 0.02% Azide

Procedure:

1. Fix embryos
   • apply appropriate fixative to embryos
   • post-fix depending on antigen
   • 3 x 5 min wash in PBST
2. Stain embryos with primary antibody
   • 30 min block
   • 2 hour stain with primary antibody diluted in block
   • 3 x wash in PBS or PBST (5' / 10' / 15')
3. Stain embryos with secondary antibody (**all steps in dark**)
   • 2 hour stain with secondary antibody (1:500 in block)
   • 3 x wash in PBS or PBST (5' / 10' / 15')
4. Mount embryos for microscopy
   • if freeze fracture, air dry slides
   • place sample in antifade for 15 minutes at room temp
   • place 6 ul on slides and apply coverslip
   • seal edges with fingernail polish

Notes:

• For freeze-fracture, apply solutions to top of slide and do washes in copland jars (35-40 mls)
• To keep primary and secondary antibody use to a minimum, place 20 ul on slide and overlay gently with a parafilm coverslip. Recycle antibody after use by pipetting off the solution
• To change media in solution fixations/stainings, centrifuge embryos at ~ 800 x g and remove supernatants
• Use PBS with 0.05 to 0.5 % detergent (Tween-20 or TritonX-100) depending on antigens
• Include DAPI @ 1 ng/ml in final wash to stain nucleic acids
• Antibody stainings can alternatively be done overnight @ 4 degrees

compiled by Chad Rappleye

Aroian Lab Protocols