Objective: to see if there is staining in the Purkinje cells or not, incubating the frozen cerebellum sections with 18-858 non-absorbed serum, or with 18-858 NF155-absorbed serum.
Protocol:
- Extract the brain and the cerebellum from the rat (healthy rat)
- Cut the brain and the cerebellum in 2 parts
- Fix the tissue by immersing all the parts in PFA 4 % (for 24 h at 4ºC)
- Put the tissues in a 30 % sucrose and 0’1% sodium azide solution (to cryoprotect them) –> incubate at 4ºC until the brain and the cerebellum are submerged in the solution
- When the tissue is in the bottom of the solution –> freeze it by using 2-methylbutane and liquid nitrogen.
- Cut one of the parts of cerebellum in frozen sections of 8 microm.
- Perform the immunohistochemistry:
- Fix with acetone (5 min)
- Wash with PBS 1x (3 x 5 min)
- Block with Blocking Goat (10 ml PBS + 0’5 ml Goat Serum + 0’2 g BSA) (30 min)
- Serum diluted in blocking goat –> samples:
- 1) 18-858 NF186-absorbed 15.11.18 (undiluted)
- 2) 18-858 HEK-absorbed 15.11.18 (undiluted)
- 3) 18-858 NF155-absorbed 15.11.18 (undiluted)
- 4) 18-858 (1/50)
- 5) Negative control (1/50)
- Wash with PBS 1x (3 x 5 min)
- Secondary antibody (1 h): GAH488 IgG diluted 1/250 in blocking goat
- Wash with PBS 1x (3 x 5 min)
- Fluoromount mounting medium
Result: no hay marcaje de las céls de Purkinje en ninguna de las muestras (sólo un poco con la muestra 4). Quizás hay que permeabilizar. Primero hay que poner a punto la immunoabsorción con el cerebelo de mono.
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