Paula’s Immunofluorescence Protocol

Day 1

Inoculate 25-50ml from a saturated starter culture. Grow overnight to OD600nm= 0.4-0.8

Day 2

Slide Preparation:

-Coat slides with 20 ul of 1 mg/ml Poly-L-Lys sol’n. Incubate for 5 min.

-Aspirate sol’n and air dry

-Wash slide in 50 ml falcon tube with ddH20 for 30 min.

-Let slide air dry

Harvest 5 OD’s of cells by vacuum filtration

Resuspend in 1 ml the same media + 1 ml 2X Fixative Solution.

Fix for 1 h at RT with gentle shaking

Harvest cells @ 3,000 rpm 5 min RT and discard supe in hazardous waste container

Wash cells 2x with 5 ml NaPi/Sorb. Buffer via vacuum filtration

Resuspend cells in 1 ml NaPi/Sorb. Buffer + 10 ul 14.3 M 2-ME

+ 10 ul of 5 mg/ml (in NaPi/Sorbitol Buffer) Zymolyase 100T

Incubate for 5-15 min @ 30ƒ C with gentle shaking to spheroplast cells

They are done when they lyse in water under the microscope.

Harvest cells 2,000 rpm, 3 min, 4ºC

Wash cells 3x GENTLY with 5 ml NaPi/Sorb. Buffer

Resuspend in 250-500 ul NaPi/Sorb. Buffer

Apply 20 ul cells to wells for 15 min.

Wash 3x with 50 ul PBS/BSA Wash Buffer

Rinse using an eppendorf repipettor and an aspirator, or a glass washing chamber, or 50 ml falcon tube.

Permeabilize cells with P Buffer for 10 min.

Wash cells 10x with 50 ul PBS/BSA Wash Buffer

Block cells 30 min with PBS/BSA Wash Buffer in dark humid chamber

Wash 5x with 50 ul PBS/BSA Wash Buffer

Incubate cells with 30 ul of 1º Ab in PBS/BSA Wash Buffer for 1 h, RT in dark moist chamber

= HA mouse Ab- 1:2000

= GFP rabbit Ab- 1:250

Wash cells 10x with 50 ul PBS/BSA Wash Buffer

Incubate cells with 30 ul of 2º Ab in PBS/BSA Wash Buffer for 1 h, RT in dark moist chamber

= GoatAntiRabbit Cy3-conjugated 1:500

= GoatAntiMouse Cy3-conjugated:500

Wash cells 7x with 50 ul PBS/BSA Wash Buffer and 3x with 50 ul PBS

Rinse using an eppendorf repipettor and an aspirator.

Mount with fluoromount G and seal with nail polish

Solutions and Buffers

2X Fixative pH 6.5 (sterile filtered)

2 % glucose 5 ml 40 % glucose

40 mM EGTA 10 ml 0.4 M EGTA

7.4 % formaldehyde 20 ml 37 % stock

in 2 X PBS 20 ml 10 X PBS

dH20 to 100 ml

NaPi/Sorbitol Buffer pH 6.6 (sterile filtered)

100 mM NaPi, pH 6.6 25ml 1 M NaPi pH 6.6

1.2 M sorbitol 75 ml 4 M Sorbitol

dH20 to 250 ml

PBS/BSA Wash Buffer (sterile filtered)

10 mg/ml BSA 1g BSA

100 ml 1 X PBS

P Buffer

10 mg/ml BSA 1 g BSA

0.5 % SDS 5 ml 10 % SDS

100 ml 1 X PBS

PBS Buffer