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:
- Collect tissues for staining.
- Fix with 1 ml of 1% PFA in a 1.5 ml tube O/N at 4 degrees.
- Wash 6 X 15 min with 1 ml of PBS.
- 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)
- 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 | |
- Incubate overnight at 4 degrees.
- Wash 3 X 40 min with 1 ml of PBS + 0.3% TX 100.
- 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) |
- Incubate overnight at 4 degrees.
- Wash 3 X 40 min with 1 ml of PBS + 0.3% TX 100.
- Add Prolong Gold + DAPI (Invitrogen; P-36931) and coverslips.