Tissue Processing

Solutions

Phosphate buffered saline (PBS)

0.1M PBS, pH 7.3

•  820 ml dH 2 0

•  160 ml 0.5M Na 2 HPO 4

•  20 ml 1M NaH 2 PO 4

•  9g NaCl

•  pH to 7.3 and filter

Avertin

Stock solution:

Avertin stock solution is photosensitive.   Prepare and store stock solution in a lightproof bottle.

•  25 grams 2-2-2 Tribromoethanol

•  15.5 ml tert-amyl alcohol

•  Stir overnight to dissolve

•  Store at room temperature

Working solution (20mg/ml):

Prepare in lightproof bottle.

•  0.5 ml avertin stock solution

•  39.5 ml PBS

•  Stir overnight to dissolve

•  Filter through 0.2 µm filter

•  Store in lightproof bottle at 4°C

Paraformaldehyde

20% PFA Stock solution:

 Prepare in fume hood.

•  200 g paraformaldehye

•  800 ml dH 2 O

•  10 ml 1N NaOH

•  Heat, stirring to 65-70°C until solution clears

•  Cool

•  Add dH 2 O to 1 liter and filter

4% Working solution:

•  20 ml 20% PFA

•  80 ml 0.1M PBS

•  pH to 7.2

Cresyl Violet Acetate

•  500 ml dH 2 O

•  1 g Sodium Acetate

•  1.5 ml Glacial Acetic Acid

•  2.5 g Cresyl violet

•  Stir until dissolved and filter.

For detailed tissue processing protocols please click on Links below:

Animal husbandry

Collection and Fixation

          E11.5 and E15.5

          P7 and P42

Embedding

          E11.5

          E15.5

          P7

          P42

Cresyl violet staining of Reference slides

Solutions

         Phosphate buffered saline (PBS)

                  - 0.1M PBS

         Avertin

                  - Stock solution

                  - Working solution

        Paraformaldehyde

                  -20% Stock solution

                  - 4% Working solution

       Cresyl Violet Acetate