March 31, 2000

Preparation of Cover slips with YTAT whole cells and YTAT cytoskeletons for IFA’s

Reagents.

Poly-L-Lysine (0.1 mg/ml) 8 ml = 160 ul of 5 mg/ml stock, 7.84 ml water.

PEME 1% NP-40 12 ml = 120 ul of NP40, 7.4 ml PEME

PBS 12 ml = filter sterilze.

PBS-0.05% tween 60 ml = 60 ml PBS, 0.3ml Tween 20

PBS, 3% (v/v) FCS, 0.05 % tween 60 ml = 1.8 ml FCS, 0.3 ml Tween20, 58 ml PBS

PBS, 3% (w/v) BSA, 0.05% tween 60 ml = 1.8g BSA, 0.3ml Tween20, 58 ml PBS

PBS, 0.5% (v/v) FCS, 0.05% tween 30 ml = 5 ml PBS 3%FCS-T, 25 ml PBS-T

PBS, 0.5% (w/v) BSA, 0.05% tween 30 ml = 5 ml PBS 5% BSA-T, 25 ml PBS-T

Protocol.

Coat each of 144 coverslips in 24 well microtitre plates with 50 ul of poly-L-lysine.

Let sit at room temp for at least 30 minutes.

Split YTAT culture (70 ml) into two equal halved, and pellet mid-log cells (YTAT1.1 @ 3.1 ee6 cells/ml) at 1000 x g.

Resuspend one tube in 4 ml PBS and Resuspend the other in 4 ml PEME 1% NP-40.

Split each of the 4 ml cultures into 3, 1.5 ml epindorf tubes (1.33 ml each).

Incubate @ room temp for 5 minutes.

Pellet @ 1000 x g in a microfuge.

Aspirate all tubes and resuspend each appropriately in 1.3 ml of either PBS or PEME 1% NP40.

Pellet @ 1000 x g for 5 minutes.

Resuspend all tubes in 1.2 ml of the appropriate buffer, and add 50 ul of cells to each cover slip. (72 cytoskeletons and 72 whole cells).

Incubate whole cells + coverslips on ice for no less than 30 minutes.

Incubate cytoskeletons + coverslips at room temp for an equal time.

Fix one half of each cell type with either Paraformaldehyde or Gluteraldehyde for no less than 30 minutes at room temperature. See diagram on following page before proceeding.

Aspirate the fixative carefully, and add 0.4 ml of the appropriate blocking solution to each.

One half of each cell preparation (36 cover slips each) will be blocked in either PBS, 3% FCS, 0.05% tween or PBS, 3% BSA, 0.05% tween.

Incubate at room temp for no less than 2 hours or indefinitely in the refridgerator. (It is recommended that after 24 hours, all cover slips be aspirated of blocking solutions and stored in PBS-T)

Add primary antibody to the appropriate cover slips in the appropriate dilutions.

After primary incubation, wash at least 3 times in PBS-T

Add secondary antibody in the appropriate dilutions (e.g. GaR FITC 1:400).

Incubate for no less than 1 hour at room temp.

Wash at least 3 times in PBS-T.

Mount each cover slip in 4 ul of Vectishield, seal with fingernail polish, and view.

GLA- fix

FCS Block

BSA Block

FCS Block

BSA Block

Plate 3 whole cells

Plate 2 whole cells

Plate 1 whole cells

PFA- fix

Diagram of Plate Format