IF on frozen sections (double IF, for 2 rat antibodies)

Immunostaining on frozen sections (double staining) (for 2 rat antibody)

Materials

Reagent name

Cat No.

Lot No.

Manufacturer

Aceton

  

270725

Sigma

Mounting Medium

H-1200

27596MK

 Vector Laboratories Inc, Burlingame

Alexa Fluor 488 goat anti-Rat IgG(H+L) (Green)

A11006

792505

Invitrogen

Alexa Fluor 594 goat anti-Rat IgG(H+L) (Red)

A11007

425995

Invitrogen

DyLight TM 488 Goat anti-rat IgG DL488

Code: 112­487-003

90908

Jackson ImmunoResearch

Methods

Day1

  1. Air dry section for 30 min at R.T.
  2. Fix in cold Aceton (-20 deg) for 15 min, air dry for a while.
  3. Wash with PBST(0.1% Tween20) 1x 3min.
  4. Draw a cycle with the Dako pen.
  5. Rinse section in PBST(0.1% Tween20) for 3min
  6. Blocking section with 5% goat serum (in 1x PBS) for 45 min at R.T.
  7. Without rinse, incubate sections with 1st primary antibody at optimized concentration (1:100~1:200 in 5% goat serum/PBS), overnight at 4 deg.

Day2

  1. Rinse in PBST 3x 5min
  2. Incubate section with 1st secondary antibody (monovalent anti-rat DL488, 1:50 in 5% goat serum/PBS), overnight at 4 deg. (or RT 1~2 hour) (Keep in the dark)

Day3

  1. Rinse in PBST 3x 5min
  2. Incubate sections with 2nd primary antibody at optimized concentration (1:100~1:200 in 5% goat serum/PBS), overnight at 4 deg. (or RT 1~2 hour) (Keep in the dark)

Day4

  1. Rinse in PBST 3x 5min
  2. Incubate section with 2nd secondary antibody (goat anti-rat red signal), for 30~45 min at R.T. (Keep in the dark)
  3. Rinse in PBST 3x 5min
  4. Mounting sections with MOUNTING MEDIUM FOR FLUORESCENCE WITH DAPI.(black plastic box on the door of 4 deg fridge.) (Keep in the dark)
  5. Observe sections under fluorescence microscope. Or store sections at 4 deg fridge for a week, or store at -20 deg fridge for longer. Store in dark, otherwise the signal will disappear soon.