Solutions:
l
4% PFA: 100 ml
4g paraformaldehyde
dissolves in 100 ml 1X PBS at 65℃
l
Permeabilisation
Solution: 50ml
serum (donkey)
5% 2.5 ml
triton X-100
0.1% 0.05 ml
sodium azide 0.05% 0.025g
l
Blocking
Solution: 20ml
serum (donkey)
5% 1ml
sodium azide 0.05% 0.01g
l
Antibody
Solution: 20ml
serum (donkey) 2% 0.4ml
sodium azide
0.05% 0.01g
l
Primary Antibody:
① Insulin 1:200 -- 5μl Insulin diluted in 1ml Antibody Solution
② Glucagon 1:200
-- 5μl glucagon diluted in 1ml Antibody Solution
l
Secondary
Antibody:
① Insulin Green 1:300-- 3μl Insulin G dilutes in 900μl Antibody Solution
② Insulin Red 1:200-- 5μl Insulin R diluted in 1ml Antibody Solution
③ Glugagon Green 1:200-- 5μl glucagon G
diluted in 1ml Antibody Solution
④ Glugagon Red 1:200-- 5μl glucagon R
diluted in 1ml Antibody Solution
Protocol:
1.
Fix cells in ice
cold 4% PFA for 7 minutes (samples can be kept for months, but make sure to parafilm the dish to avoid drying out)
2.
Wash 2x with PBS
3.
Leave some PBS in
the bottom of the dish and, using tissue paper, soak up PBS around the rim,
leaving a drop of PBS in the middle (where the cells are)
4.
Draw around this
part with a PAP pen (to leave a hydrophobic barrier for small volume — one drop
— around 20μl - 50μl
5.
Add Permeabilisation Solution within the PAP
circle for 10 minutes on ice
6.
Wash 1x with PBS
7.
Add Blocking
Solution for 1hr at room temperature
8.
Wash 1x with PBS
9.
Add Primary
Antibody for 2 hr at room temperature or overnight at 4℃
10. Wash 2x with PBS
11. Add Secondary Antibody for 1 hr at room
temperature (in the dark)
12. Wash 3x to 5x with PBS