Jacobs: Protocol Immunostaining for TRPV4 and primary cilia
Immunostaining for TRPV4 and primary cilia
- Samples: Primary osteocytes serum starved (0.5% FBS) for 2 days, growing on 24-well glass-bottom dishes treated with collagen
- Aluminum Foil
- Pipets/Pipet aid
- Pipet tips/pipetters
- Waste beaker
- 50 mL centrifuge tube (“formaldehyde waste”)
- 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)
- Wash cells 2X with 4 degree PBS (500 ul per wash, use pipetman) – remove all PBS from around slide after final wash
- 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
- Wash cells 2X with PBS – place waste in the “formaldehyde waste” tube
- Permeablize cells: apply 200 uL of cold 0.1% Triton X-100 solution for 4 min.
- Wash cells 2X with PBS
- Nonspecific blocking: 1% BSA in PBS for 1 hour room temp (200 uL per well)
- 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)
- Remove block.
- Incubate with solution of primary antibody in 1%BSA/PBS for 2 hours at RT (200 ul)
- In the dark, make up secondary antibodies in 1%BSA/PBS, and keep dark.
- 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
- 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)
- Wash 3X 1%BSA in PBS. Immediately remove first wash then ~5 min per wash.
- 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.
- Make up DAPI: for 1 ml 1% BSA PBS, add 2 ul DAPI for 1 ug/ml
- Wash 3X with 1% BSA PBS. Immediately remove first wash then ~5 min per wash.
- Nuclear stain: DAPI, 1 ug/ml, incubate 40 min. @ RT (200 μL)
- Take out Prolong Gold Antifade reagent to defrost
- Wash coverslip area 3X with 1% BSA PBS
- 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
- Seal edges of dish using parafilm.
- Image using confocal microscope. Have to use multiphoton for imaging DAPI. Take images of entire cell, and zoomed into primary cilia.
- History: JCC, last updated 3/1/11