1. Fixation:
3% PFA in 0.1M Buffer with 0.1% GA and 4% sucrose
2. Wash:
4x15 min. with correlative buffer containing 0.1M Sucrose @ 4C
1x30 min. with correlative buffer containing 0.1M Sucrose and 0.1M Glycine @4C
3. Dehydration with METOH:
30% 1x15 min. @4C
50% 1x15 min. @4C
70% 1x15 min. @-10C
85% 1x15 min. @-20C
95% 1x15 min. @-20C
100% 3x15 min @-30C
6. Infiltration with Lowicryl LK4M:
METOH:LK4M = 1:1 for 1 hr. @-30C
METOH:LK4M = 1:2 for 1 hr. @-30C
METOH:LK4M = 1:3 for 1 hr. @-30C
Pure LK4M 1 hr. @-30C
Pure LK4M overnight @-20C
7. Embedment:
Embed into Beem Capsules and cure under UV in a CO2 environment @-35C for 36 hr. and under UV in a CO2 environment for 12 hr. @ RT.
Showing posts with label IEM. Show all posts
Showing posts with label IEM. Show all posts
Thursday, June 11, 2009
Postembedding Immunolabeling
Postembedding Immunolabeling
1. Cut sections (>100 nm thick) on formvar coated Ni grids
2. Labeling on drops
a. 1x5 min. with 0.1MPBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Triton x-100
iii. 0.05% Tween 20
b. 2 hr. @ RT and @4C overnight with Antibody in 0.01M PBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Tween 20
c. Wash 3x1 min. on drops of 0.01M PBS
d. 1 Hr. @RT Incubation with secondary antibody conjugated with Au labels in 0.1M PBS containing
i. 1% BSA
ii. 0.05% Tween 20
e. Wash 3x1 min. on drops of 0.01M PBS
f. Allow grids to dry
3. Counter stain
a. 3% UA 4 min.
b. Lead citrate <1min.
1. Cut sections (>100 nm thick) on formvar coated Ni grids
2. Labeling on drops
a. 1x5 min. with 0.1MPBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Triton x-100
iii. 0.05% Tween 20
b. 2 hr. @ RT and @4C overnight with Antibody in 0.01M PBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Tween 20
c. Wash 3x1 min. on drops of 0.01M PBS
d. 1 Hr. @RT Incubation with secondary antibody conjugated with Au labels in 0.1M PBS containing
i. 1% BSA
ii. 0.05% Tween 20
e. Wash 3x1 min. on drops of 0.01M PBS
f. Allow grids to dry
3. Counter stain
a. 3% UA 4 min.
b. Lead citrate <1min.
Wednesday, June 10, 2009
Processing tissue for Immuno Electron Microscopy
Again buffers and fixative percentages can vary from sample to sample but here's my protocol:
01) Fixation 2hrs. @RT and o/n @4C
02) Wash with buffer containing 4% sucrose 4x15 min. @4C
03) Wash with buffer containing 4% sucrose and 0.1M Glycine 1x30 min. @4C
04) Dehydration with Methanol:
30% 1x15 min. @4C
50% 1x15 min. @4C
70% 1x15 min. @-10C
85% 1x15 min. @-20C
95% 1x15 min. @-20C
100% 3x15 min. @-30C
05) Infiltration with Lowicryl LK4M
Methanol:LK4M = 1:1 1 hr. @-30C
Methanol:LK4M = 1:2 1 hr. @-30C
Methanol:LK4M = 1:3 1 hr. @-30C
Pure LK4M 1 hour @-30C
Pure LK4M o/n @-20C
06) Embed in Beem capsules @-35C with dry nitrogen bubbling in Lowicryl
07) Cure under UV light in a dry ice environment @-35C 36hrs. and @RT for 12 hrs
01) Fixation 2hrs. @RT and o/n @4C
02) Wash with buffer containing 4% sucrose 4x15 min. @4C
03) Wash with buffer containing 4% sucrose and 0.1M Glycine 1x30 min. @4C
04) Dehydration with Methanol:
30% 1x15 min. @4C
50% 1x15 min. @4C
70% 1x15 min. @-10C
85% 1x15 min. @-20C
95% 1x15 min. @-20C
100% 3x15 min. @-30C
05) Infiltration with Lowicryl LK4M
Methanol:LK4M = 1:1 1 hr. @-30C
Methanol:LK4M = 1:2 1 hr. @-30C
Methanol:LK4M = 1:3 1 hr. @-30C
Pure LK4M 1 hour @-30C
Pure LK4M o/n @-20C
06) Embed in Beem capsules @-35C with dry nitrogen bubbling in Lowicryl
07) Cure under UV light in a dry ice environment @-35C 36hrs. and @RT for 12 hrs
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