Imaging and histology prep

Goals:

a)        Training on CCAM confocal microscopy

b)       Test eBioscience Perm (0.1% saponin) kit for preparing confocal slides

c)        Test mounting of Ramos to coverslip versus ibidi plate

Experimental Conditions::

A)       Ramos Cell line (Human B cells) (1X)

B)       Stain for tubulin (1X)

C)      Mounting option (2X)

D)      Replicates (2X)

Total n=4

Prepare O/N::

1. Poly-L-lysine coated ibidi plate & coverslip: incubate in 0.01% poly-L-lysine @4C for O/N. (Can reuse excess)

Protocol::

1. Prepare RT permealization buffer + BSA +/- 1o Ab (must be freshly prepared)

2. EXTRACELLULAR STAIN

   1. 0.5-1x10^6 cells

   2. 100uL of stain in PBS+2%FBS OR BSA

   3. Incubate on ICE for 15-20mins (NO WASH)

3. FIXATION

   1. Add COLD PFA to 1% (1uL into 100uL)

   2. DO NOT VORTEX, GENTLY PIPETTE 2-5times

   3. Incubate in the DARK on ICE for 10mins

   4. Centrifuge 500xg 4C 5mins, discard supernatant

4. PERMEABILIZE

   1. Wash-perm #1: Resuspend in 200uL RT 1X permeabilization buffer + BSA, pipette ~10-15times (avoid bubbles), Centrifuge spin 500xg RT 5mins, discard supernatant

   2. Wash-perm #2: repeat (4a)

5. 1o STAIN

   1. Resuspend in 50uL RT 1o Ab in permeabilization buffer + BSA

   2. Incubate in DARK at RT for ≥30mins

   3. Centrifuge spin 500xg RT 5mins, discard supernatant

   4. Wash-PBS #1: Resuspend in 100uL PBS, Centrifuge spin 500xg RT 5mins, discard supernatant

   5. (Optional) Wash-PBS #2: repeat (5d)

6. 2o STAIN

   1. Resuspend in 50uL RT 2o Ab in PBS

   2. Incubate in DARK at RT for ≥30mins

   3. Wash-PBS #1: Resuspend in 100uL PBS, Centrifuge spin 500xg RT 5mins, discard supernatant

   4. Wash-PBS #2: repeat (6c)

   5. Resuspend in PBS

7. MOUNT

   1. Test mounting method:

      1. Option A) Transfer <500uL to poly-L-lysine coated ibidi plate, centrifuge 500xg 4C 5min. Samples ready for analysis 

      2. Option B) Transfer to 12-well plate w/ poly-L-lysing coated coverslip at bottom, centrifuge 500xg 4C 5mins. Remove supernatant. Place excess mounting solution onto a slide. Place coverslip onto mounting solution+slide, CELLS SIDE DOWN INTO MOUNTING. Soak up excess mounting solution. Samples ready for analysis