• Warm stretcher, media, fix in 37 degree oven
• Prep fresh TPBS/BSA, 1%, 5% NGS solutions
• Calculate Ab dilutions, collect primaries
• Fill stretching chamber halfway with 37º culture media, keep strip moist
• Mount silastic strips in clamps, measure resting length
• Raise level of media so that cells are covered
• Stretch to desired length
• When ready to fix, drain media, dry completely with filter paper
• Dry ends of strips with filter paper and create a grease barrier
• Fix for10 min with 45 uL warm 3% paraformaldehyde/PBS
• Add 45 uL 0.2% Triton-X100 in PBS for 10 min
• Rinse 3x with PBS

Immunofluorescence

• Add TPBS/BSA/5%NGS
• Allow this to sit in a humidity chamber for 20 minutes
• Add primary antibody in 1%NGS/TPBS/BSA for 1 hr at 37degreesC.
• Prep secondary antibody during incubation
• Wash 3 X 10 min each with TPBS/BSA buffer
• Add Secondary antibody (in PBS) and incubate for1 hr at 37°C
• Wash 3 X 10 min each with TPBS/BSA buffer
• Mount slides in Vectashield (pH 8.2).

STOCK SOLUTIONS

(1) 0.2 M Na/K phosphate PH 7.3 250 ml

Solution A 0.2 M Na2HPO4 Sigma #S-0876 Anhydrous FW 142.0 g
0.2 M Na2HPO4 = 7.1 g made up to 250 ml dH20

Solution B: 0.2 M KH2P04 Sigma #P-5379 Anhydrous FW 136.1
0.2 M KH2P04 = 6.805 g made up to 250 ml dH20

For 0.2 M Na/K phosphate buffer at pH 7.3 start with 150 mL 0.2 M Na2HP04 (Solution A) and titrate to pH 7.3 with 0.2 M KH2P04 (Solution B)

(2) 1.5 M KCl Sigma P-4504 FW = 74.55

1 M = 74.55 g in 1000 ml
1.5 M = 111.825 g in 1000 ml
1.5 M = 27.96 g made up to 250 ml w/ dH20

(3) 1.5 M NaCl Sigma S-9625 FW = 58.44)

1.5 M = 21.92 g made up to 250 ml w/ dH20 

WORKING SOLUTIONS

(1) PBS (phosphate buffered saline) pH 7.3

Vol. to add Stock Final Conc. Final Vol.

50.00 ml 1.5 M NaCl 150.0 mM

10.00 ml 0.2 M Na/K PO4 4.0 mM pH 7.3
1.66 ml 1.5 M KCl 5.0 mM
__________
500 ml

Bring to about 200 ml w/ dH20 then adjust pH to 7.3.
Bring up to final volume. 

(2) Fluorescence fixative (3% paraformaldehyde in PBS) pH 7.3
Vol. to add Stock Final Conc. Final Vol.
7.5 g Paraf. solid 3%

Add the 7.5g paraformaldehyde to approx. 150 ml dH20 in a 250 ml beaker with a stirring bar. Place on a hot plate in the fume hood and carefully place a thermometer into the solution. Bring the temp. to 60 oC. Clear solution with 0.1 M NaOH added drop by drop. Check the pH paper and do not let it get above 7.3. When solution is clear, take off heat and cool to room temperature. Add the following constituents to complete the fixative.)

25.00 ml 1.5 M NaCl 150.0 mM 
5.00 ml 0.2 M Na/K PO4 4.0 mM pH 7.3
0.83 ml 1.5 M KCl 5.0 mM
__________
250 ml
Bring to about 200 ml w/ dH20 then adjust pH to 7.3.
Bring to volume.

Working solutions for immunofluorescence:

(1) TPBS/0.1% BSA buffer
50 microL of Tween

0.1 g BSA (Sigma # A 9647)

0.1% in 100 mL PBS, adjust pH to 7.3, 

Antibody Buffers:

for ALL:

(1) 5% normal Goat serum (NGS) /Tween-20/PBS/BSA buffer

50 uL NGS in 950 μL Tween-20/PBS/BSA buffer

(2) 1% NGS /Tween-20/PBS/BSA buffer

40 uL NGS in 3.96 ml Tween-20/PBS/BSA buffer