Immunohistochemistry (cultured cells/monolayers)

Contributed by Luke Hammond, QBI, The University of Queensland, Australia

Immunohistochemistry (cultured cells and monolayers)

This protocol outlines a general method for immunohistochemical staining and can be adjusted to suit the antibodies used.


Fixative Use 4% PFA (sometimes kept as frozen 8% stock – add 2xPBS buffer to make up to 4%) Blocking Buffer 0.5% BSA/PBS (e.g. 0.5g BSA in 100ml PBS) (% = g/100ml) Permeabilisation Buffer 0.1% TX-100 in PBS (e.g. 10% Tx-100 stock then 200µl in 20ml PBS)


  • Fix cells: 4% paraformaldehyde in PBS, pH7.4 for 30-90 mins RT
  • Wash gently x3 PBS
  • Permeabilise cells with 0.1% TX-100 in PBS for 5mins
  • Wash 2x in PBS
  • Block for 10min in 0.5% BSA/PBS (but can be as long as you like)
  • While blocking should make up primary antibody, dilute antibody in BSA/PBS, need 25-40ul/coverslip

E.g. Antibody 1:50 = 2ul in 100ul BSA/PBS

  • Aliquot onto parafilm with sufficient space between coverslips
  • Pick up coverslip and dry edges gently (touch corner to kimwipe) and place coverslip cells down onto antibody drop. Incubate for 60-90min RT
  • Flip coverslip so cells face up and wash gently with BSA/PBS x 3
  • Prepare secondary antibody (25-45ul/coverslip) and incubate for 45-60min

Many 2° Ab are used 1:400 (e.g. 1ul per 400ul) in BSA/PBS

  • Wash x3 BSA/PBS
  • Wash x3 PBS

This method is based, with permission, on an original protocol available here.