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Day1

Stock Solutions

  • PBS
  • 4% PFA
  • 1% Tween-20 in PBS
  • 1% Triton X-100 in PBS
  • 5% BSA in PBS
  • antibodies

Solutions to be used

  • PBS, used 6x
  • Permeabilization Buffer (Day1, 0.1% Triton X-100, 1% BSA in PBS), used 1x
  • Blocking/primary antibody incubation Buffer (Day1, 0.05% Tween-20, 1% BSA in PBS), used 2x
  • Washing Buffer/secondary antibody incubation buffer (Day2, 0.05% Tween-20 in PBS), used 3x
  • Primary antibodies, used 1x
  • Secondary antibodies, used 1x

Volumes

  • 1 well is 300$\mu$L volume (for washing and antibody incubation)

Cells fixation

  • move to chemical cabinet in genomic room
  • aspirate media
  • wash with PBS
  • fix with 4% PFA for 10 min. at RT
  • aspirate
  • wash with PBS
  • aspirate
  • add PBS for storage
  • store at 4$^\circ$C

Incubation with primary antibodies

  • Permeabilze with 0.1% Triton X-100 in PBS for 15 minutes at RT.
  • Remove permeabilization solution.
  • Wash 1 x with PBS.
  • Add blocking solution (1% BSA in 0.05% Tween-20 PBS) and incubate for 30 minutes.
  • Aspirate the blocking solution.
  • Add diluted antibodies in (1% BSA in 0.05% Tween-20 PBS) overnight at 4$^\circ$C

Day 2

Solutions to prepare

  • 0.05% Tween 20 in PBS (for 3 washes and antibody incubation)
  • Solution of secondary antibody in 0.05% Tween 20 in PBS (if there are more secondary antibodies mix them together)
  • DAPI in PBS (1:5000) (make 10mL stock)
  • PBS
  • Mowiol

Process

  • Aspirate primary antibody.
  • Wash with 0.05% Tween 20 in PBS (3x)
  • Prepare secondary antibody(ies) solution
  • Add secondary antibody to the wells
  • Incubate for 1h with secondary antibody
  • Aspirate
  • Wash 1x with PBS
  • Add DAPI working solution to stain nuclei
  • Incubate 5min
  • Aspirate
  • Wash 3x 5 min with PBS
  • Check under microscope
  • Mount in Mowiol
  • Store at 4$^\circ$C in dark

protocol END

To Calculate:

Given you have 4 wells

  1. How much permeabilization buffer to prepare and how
  • 5% BSA:
  • 1% TX-100:
  • PBS:
  1. How much Blocking/primary antibody incubation buffer prepare and how
  • 5% BSA:
  • 1% TW-20:
  • PBS:
  1. Primary antibodies:
  • BNP antibody:
  • SarcAct antibody:
  • Blocking Buffer:
  1. Secondary antibodies:
  • anti-mouse A488:
  • anti-rabbit A555:
  • Washing Buffer/secondary antibody incubation buffer: