Jacobs: Protocol Immunostaining for TRPV4 and primary cilia

Immunostaining for TRPV4 and primary cilia
JCC 2/24/2011

Materials

 * Samples: Primary osteocytes serum starved (0.5% FBS) for 2 days, growing on 24-well glass-bottom dishes treated with collagen
 * Kimwipes
 * Aluminum Foil
 * Pipets/Pipet aid
 * Pipet tips/pipetters
 * Aspirator
 * Timer
 * Waste beaker
 * 50 mL centrifuge tube (“formaldehyde waste”)
 * Markers
 * Gloves
 * parafilm
 * Phosphate-buffered saline (PBS), pH 7.4
 * 10% formalin
 * 0.1 % Triton X-100 solution (in PBS)
 * Mounting media- Prolong Gold Antifade Reagent (Invitrogen, P36930)
 * Primary Blocking Solution: PBS + 1% (w/v) BSA (Sigma A2058, IgG free, be sure to use correct bottle, smaller one) – stir on plate to dissolve, then filter
 * Primary antibody: acetylated alpha tubulin (Abcam ab24610, mouse, 1:1000), TRPV4 (Heller antiserum, rabbit, 1:500, Cuajungco J Biol Chem 2006)
 * Secondary antibody- make up in the dark (Invitrogen goat anti rabbit, Alexa Fluor 488 A11008; Invitrogen goat anti mouse, Alexa Fluor 647 A21236 OR Invitrogen goat anti mouse, Alexa Fluor 568 A11031; 1:200 for each)
 * DAPI (0.5 mg/ml)

Procedure

 * 1) Wash cells 2X with 4 degree PBS (500 ul per wash, use pipetman) – remove all PBS from around slide after final wash
 * 2) Fix cells: apply 200 ul of 10% formalin solution on top of slide for 10 min. @ RT. Note: formaldehyde is toxic; do in fume hood, with gloves, avoid contact with skin, eyes, etc.; dispose of as hazardous waste in tube, do not pour down sink
 * 3) Wash cells 2X with PBS – place waste in the “formaldehyde waste” tube
 * 4) Permeablize cells: apply 200 uL of cold 0.1% Triton X-100 solution for 4 min.
 * 5) Wash cells 2X with PBS
 * 6) Nonspecific blocking: 1% BSA in PBS for 1 hour room temp (200 uL per well)
 * 7) Make up primary antibodies in 1%BSA/PBS:  Acetylated alpha tubulin and TRPV4- For every 1 ml of BSA/PBS, add 1 ul each of acetylated alpha tubulin and 2 ul of TRPV4 antibodies (need 200 ul of solution per sample, make extra)
 * 8) Remove block.
 * 9) Incubate with solution of primary antibody in 1%BSA/PBS for 2 hours at RT (200 ul)
 * 10) In the dark, make up secondary antibodies in 1%BSA/PBS, and keep dark.
 * 11) Alexa Fluor 647 (previously used): For every 1 ml of BSA/PBS, add 5 ul each of goat-anti-rabbit and 5 ul of goat-anti-mouse 647 (need 200 ul per sample)  OR
 * 12) Alexa Fluor 568: For every 1 ml of BSA/PBS, add 5 ul each of goat-anti-rabbit and 5 ul of goat-anti-mouse 568 (need 200 ul per sample)
 * 13) Wash 3X 1%BSA in PBS.  Immediately remove first wash then ~5 min per wash.
 * 14) Incubate 1:200 of secondary antibody 1%BSA/PBS for 1hour at RT in the dark (200 ul) Note: Keep samples in the dark from now on.
 * 15) Make up DAPI: for 1 ml 1% BSA PBS, add 2 ul DAPI for 1 ug/ml
 * 16) Wash 3X with 1% BSA PBS. Immediately remove first wash then ~5 min per wash.
 * 17) Nuclear stain: DAPI, 1 ug/ml, incubate 40 min. @ RT  (200 μL)
 * 18) Take out Prolong Gold Antifade reagent to defrost
 * 19) Wash coverslip area 3X with 1% BSA PBS
 * 20) Remove the Blocking Solution and add Prolong Gold Antifade Reagent mounting medium (25uL, or enough to cover, per glass area) to slide.  Tip: Suction ~100 ul, and then add one drop per well
 * 21) Seal edges of dish using parafilm.
 * 22) Image using confocal microscope.  Have to use multiphoton for imaging DAPI.  Take images of entire cell, and zoomed into primary cilia.  If not imaging immediately, store in the dark at 4C for ~1 week.  Image as soon as possible though.


 * History: JCC, last updated 3/14/11