- Transfer tissue to 24 well nets
- Rinse out cryo w/ 50mMTBS7.2 X # rinses, time of each rinse
- LTBS= ?mL 50mMTBS7.2 + ?µl 25%T + ?mL NDS
- 1˚-
- 1 aliquot= ?µl
- 1:?k= take ?µl
- add ?ml LTBS= ?ml 1:?k
- use ?mL for 1:?k
- 1:?k= take ?mL 1:?k
- add ?mL LTBS= ?ml 1:?k
- use ?mL for 1:?k
- 1:?k= take ?mL 1:?k
- add ?mL LTBS= ?mL 1:?k
- Pre-incubate/Preabsorb for ? min
- Transfer to 1˚ -> 4˚ @ time
Date
- Rinse out 1˚ @ time
- total time in 1˚
- 50mMTBS7.2 X # rinses, time of each rinse
- Sort sections for secondary reaction
- LTBS= ?mL 50mMTBS7.2 + ?µl 25%T + ?mL NDS
- 2˚-
- name the secondary being used at what concentration
- calculate the dilution of the secondary
- Pre-incubate/preabsorb for ? min
- Transfer to 2˚ for ? time
- Rinse out 2˚ at @ time
- total time in 2˚
- 50mMTBS X # rinses, time of each rinse
- 3˚-
- name the fluor label being used at what concentration
- calculate the dilution of the label in 50mMTBS7.2
- Transfer to 3˚ at ? time
- Rinse out 3˚ at ? time
- total time in 3˚
- 50mMTBS X # rinses, time of each rinse
- Transfer sections to 6-well trays @ time to await mounting
To this you should add a place for the tissue used, including series and section info, a place for a title--a one line description of the immuno being run (e.g., AChE, NOS, & TH Flourescence Dilution Curves). Also add something like Chromagens/fluorochromes _________. For LTBS note the final concentration of Triton is 0.1% and NDS is 3%. Note that 3º is either a streptavidin conjugate or an anti-FITC, and either 3º could be conjugated to HRP or a fluorochrome....
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