IF on frozen sections (double IF)

Immunostaining on frozen sections (double staining)

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

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

A11008

913909

Invitrogen

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

A11012

57911A

Invitrogen

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

A11011

898287

Invitrogen

Alexa Fluor 568 goat anti-mouse IgG(H+L) (Red)

A11004

629102

Invitrogen

Alexa Fluor 546 goat anti-chicken IgG(H+L) (Red)

A11040

682609

Invitrogen

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

A11080

459560

Invitrogen

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 30~45 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 (goat anti-rat/rabbit/mouse; red or green signal), for 30~45 min at R.T.
  3. Rinse in PBST 3x 5min
  4. Re-blocking section with 5% goat serum (in 1x PBS) for 45 min at R.T.
  5. Without rinse, incubate sections with 2nd primary antibody at optimized concentration (1:100~1:200 in 5% goat serum/PBS), overnight at 4 deg. (Keep in the dark)

Day3

  1. Rinse in PBST 3x 5min
  2. Incubate section with 2nd secondary antibody(goat anti-rat/rabbit/mouse; green or 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, or the signal will disappear soon.

Note: if you only need to do ONE marker, then just stop at step 9 and continue step 15.