Whole-Mount Staining

Date:

Purpose:

Check if used Tissue for staining
Collect ear and pull apart two layers of skin.  Use the top (outer layer) for staining.
Collect yolk sacs from E_____ embryos.
Collect diaphragm from adult mouse.
Collect mesentery from mouse.
Collect trachea from mouse.

Mice:

Protocol:

  1.  Collect tissues for staining.

 

  1. Fix with 1 ml of 1% PFA in a 1.5 ml tube O/N at 4 degrees.
  2. Wash 6 X 15 min with 1 ml of PBS.
  3. Block overnight at 4 degrees with 1 ml of PBS + 0.3% TX 100 + 20% Aquablock. (Aquablock can be substituted with your lab’s standard blocking reagent)
  4. Dilute primary antibodies in 1 ml of PBS + 0.3% TX 100.
Check if used Primary Antibody Company and catalog
Goat anti-Lyve-1 (1:1000) R&D Systems; AF2125
Rat anti-CD31 (MEC 13.3) (1:500) BD Pharmigen; 553370
Hamster anti-podoplanin (1:1000) Abcam; ab11936
Rabbit anti-Prox1 (1:1000) Abcam; ab101851

 

  1. Incubate overnight at 4 degrees.
  2. Wash 3 X 40 min with 1 ml of PBS + 0.3% TX 100.
  3. Dilute secondary antibodies in 1 ml of PBS + 0.3% TX 100.
Check if used Secondary antibody (circle if Cy3 or FITC). Add at 1:500.
FITC   or   Cy3   Donkey anti-goat (Jackson ImmunoResearch)
FITC   or   Cy3   Donkey anti-rabbit (Jackson ImmunoResearch)
FITC   or   Cy3   Donkey anti-hamster (Jackson ImmunoResearch)
FITC   or   Cy3   Donkey anti-rat (Jackson ImmunoResearch)
FITC   or   Cy3   Donkey anti-Chicken (Jackson ImmunoResearch)
Cy3 conjugated Mouse anti-smooth muscle actin (Sigma; C6198)

 

  1. Incubate overnight at 4 degrees.
  2. Wash 3 X 40 min with 1 ml of PBS + 0.3% TX 100.
  3. Add Prolong Gold + DAPI (Invitrogen; P-36931) and coverslips.