Shreffler:Confocal Protocols

Overview
Collection of protocols used by Alex for the basophil imaging project.

Materials

 * 12 well tissue culture plate
 * round cover glasses (i.e. "slides")
 * 70% Ethanol (aq)
 * 1X Poly-L-Lysine (1:10, Poly-L-Lysine:dH2O)

Procedure

 * 1) Wash circular cover glasses in a small petri dish filled with 70% ethanol. Transfer cover glasses individually to the wells of culture plate with clean forceps and allow to air dry.
 * 2) Under the hood apply 100 μL of 1X Poly-L-Lysine solution to center of each slide.
 * 3) Let slides stand for 20 minutes before aspirating off excess Poly-L-Lysine solution.  Let slides air dry under hood.  Slides can be stored at room temperature until ready to use.

Procedure
fMLP


 * For first dilution (1:100), add 1 μL fMLP to 99 μL RPMI.
 * Second dilution, take 3 μL from this dilution of fMLP and add it to a separate tube containing 297 μL RPMI (1:100).
 * Add 250 μL of the second solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C.

RPMI: Unstimulated Control

 * Add 250 μL RPMI to 250 μL basophil cell solution.
 * Incubate for 30 minutes at 37 °C.

Anti-IgE


 * Add 0.6 μL Anti-IgE to 300 μL RPMI
 * Add 250 μL of this solution to 250 μL of basophil cell solution and incubate for 30 minutes at 37 °C.

Materials

 * 30 ml PBS (Phosphate Buffered Saline)
 * 30 ml 4% Paraformaldehyde in PBS (PFA-PBS); made from 5 ml stock PFA in 25 ml PBS

Procedure

 * 1) Once basophil cells are extracted from PBMC and stimulated, spin cells down at 300g for 7 minutes.
 * 2) Aspirate RPMI and add 50ŒºL PBS to each tube.
 * 3) At 4 °C, apply ~50 μL basophil-PBS solution to center of  Poly-L-Lysine-coated slides.
 * 4) Allow solution to sit for 15-20 minutes, undisturbed at 4 °C, so that cells can settle onto slide.
 * 5) Check with microscope to ensure cells adequately settled onto slide.
 * 6) Aspirate liquid from surface of slide, and wash each slide with 1mL PBS.  To minimize cell loss allow PBS to run down the side of the well while washing.
 * 7) Aspirate and fix cells with 1 mL 4% PFA-PBS.  Incubate at 4 °C in the dark for 15 minutes.
 * 8) Keep Cells on ice and in the dark until subsequent procedures are carried out.

Materials

 * 15 mL PBS
 * 100 mL 0.1% Triton X-100 PBS (TxPBS); made from 1 mL stock Triton X-100 in 100 mL PBS
 * 15 mL Stain Buffer (1% bovine albumin in 2 mM EDTA in PBS)

Procedure

 * 1) Wash slides once with 1 mL PBS per well.
 * 2) Wash slides 3X with TxPBS.  Each wash should incubate for 5 minutes between washes.
 * 3) Block non-specific binding by adding 1mL Staining Buffer. Incubate for 30 minutes at room temperature in the dark.
 * 4) Prepare Ab solution and apply as described in separate procedure.

Procedure

 * 1) Overnight Staining with goat anti-CD203c (1:100 from 200 μg/mL stock); rabbit anti-CD107a (1:400 from 200 μg/mL) in staining buffer
 * 2) Next morning, wash 3 times for 5 minutes with TxPBS
 * 3) Stain with biotinylated donkey anti-goat (1:1000), incubate 1 hour.
 * 4) Wash 3 times for 5 minutes with TxPBS
 * 5) ; Prepare CD63 Ab: For every 0.5 mL (1 SLIDE) add 4 μL mouse anti-CD63 (1:125) in 15 uL staining buffer, add 8 μL Zenon Alexa 594. Incubate 10 min at RT, add 8 μL Zenon blocking reagent. Incubate 10 min at RT.
 * 6) Stain with anti-CD63 cocktail, with 1:125 dilution of strepavidin Alexa 488 (for CD203c), and 1:400 donkey anti-rabbit 647 (for CD107a).  Incubate for 1 hour.
 * 7) Wash 3 times for 5 minutes with TxPBS
 * 8) Fix with 4% PFA.  Incubate for 15 minutes.
 * 9) Aspirate excess PFA, and mount slides with DAPI Vectashield mounting medium.  Store at 4 °C in the dark until acquisition.

Discussion
discuss this protocol

Contact

 * Who has experience with this protocol?
 * Email Alex Praslick through OpenWetWare