-
Categories
-
Pharmaceutical Intermediates
-
Active Pharmaceutical Ingredients
-
Food Additives
- Industrial Coatings
- Agrochemicals
- Dyes and Pigments
- Surfactant
- Flavors and Fragrances
- Chemical Reagents
- Catalyst and Auxiliary
- Natural Products
- Inorganic Chemistry
-
Organic Chemistry
-
Biochemical Engineering
- Analytical Chemistry
- Cosmetic Ingredient
-
Pharmaceutical Intermediates
Promotion
ECHEMI Mall
Wholesale
Weekly Price
Exhibition
News
-
Trade Service
Protocol: Immunofluorescence / confocal microscopy
B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:
1. wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
5. wash 1x PBS/BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
7. wash 1x PBS/BSA.
8. 1 ° diluted in PBS/BSA 60 min RT; 100 l per tube or section.
9. wash 3x PBS/BSA.
10. Block 15 min 5% NGS in PBS/BSA.
11. wash 1x PBS/BSA.
12. 2 ° diluted in PBS/BSA 30 min RT; 100 l per tube or section.
13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash
**This is a "light" fix due to the sensitivity of the antigen(s). Cells should be imaged as soon as possible.
Another fix for cryostat sections:
(Unfixed tissue embedded in OCT and frozen)
1. Fix slides with sections (using VWR Superfrost Plus slides) in -20 ° acetone for 10 min.
2. wash 3x PBS/0.5% BSA.
3. Block and label as above #6-13 except use 0.5% BSA in wash with PBS. Use ProLong antifade to coverslip.
Notes:
¨ When looking at B cells (they are relatively fragile) I have tried to support the coverslip with EM grids on two sides, 12 m m diameter latex beads from Sigma (LB-120) mixed with the final cell suspension in antifade medium, and have tried #1 coverslips instead of #1.5. Using #1 coverslips has given me the fewest smashed cells. I now use an inverted scope and pipet the cells onto coverslip chamber slides (Lab-Tek #136439) in antifade medium so no worries about flattened, smashed cells.
smashed by coverglass | looking pretty good |
¨ To look at cytoskeleton I have fixed adherant cells on chambered coverslip slides and cryostat sections with ice cold methanol:acetone 1:1 for 10 minutes, then in ice cold ethanol:acetic acid 95:5 for 10 minutes, then wash in saline. Block and label as above steps 6-13.
keratin in cultured human mesangial cell | keratin in cryostat section kidney proximal tubule |
¨ Vectashield (Vector Laboratories H-1000 and H-1200 with DAPI) antifade medium gives better antifade protection than SlowFade Light but is hard on the cells; I see lots of membrane blebbing on both fixed B cells in suspension and on fixed adherent melanoma cell lines. Sometimes the problem is worse than