Protocols

We recommend to use the following protocols with our antibodies

Our antibodies are equipped with an add-on technology that enables a stronger signal, thus making them more sensitive but also slightly bulkier.

To allow the molecules into the cells, customers are advised to follow our protocols published on this page. For both intracellular and extracellular staining, it is advised to run all protocols in parallel to see which gives the best signal.

Most protocols uses SDS to denature and relax targets and to reveal epitopes, thus increasing antibody binding.

Immunofluorescence - Frozen tissue sections or cultured cells

Protocol 1: Fix with PFA, Permeabilise with Triton X-100 and SDS (Recommended)

  1. Fix specimen with 4% Paraformaldehyde for 10 minutes (Cover at depth of 2-3mm).
  2. Wash 3x times with 0.1%SDS in PBS for 5 min each.
  3. Block specimen with Antigen retrieval/Permeabilisation solution (0.1% SDS / 0.1% Triton X-100 / 1% BSA in PBS), Incubate for 30 minutes.
  4. Wash 3x with PBS for 5 min each.
  5. Apply Antibody (1:200 in PBS) for 1 hour in the dark.
  6. Wash 3x in PBS for 5 min each in the dark.
  7. Mount specimen for imaging.

Protocol 2: Fix with PFA, Permeabilise with Methanol and SDS

  1. Fix specimen with 4% Paraformaldehyde for 10 minutes (Cover at depth of 2-3mm).
  2. Permeabilise with ice-cold (-20°C) 100% Methanol for 15 min on ice or 4°C (Cover at depth of 2-3mm).
  3. Wash 3x times with 0.1%SDS in PBS for 5 min each.
  4. Block specimen with 0.1% SDS/1% BSA in PBS for 30min.
  5. Wash 3x with PBS for 5 min.
  6. Apply Antibody (1:200 in PBS) for 1 hour in the dark.
  7. Wash 3x in PBS for 5 min each in the dark.
  8. Mount specimen for imaging.

Protocol 3: Fix/permeabilise with Methanol, followed by PFA and antigen retrieval with SDS

  1. Fix/Permeabilise specimen with 1ml ice-cold (-20°C) 100% Methanol for 15 min on ice or 4°C (Cover at depth of 2-3mm).
  2. Wash 1x times with PBS for 5 min each.
  3. Fix specimen with 4% Paraformaldehyde for 10 minutes (Cover at depth of 2-3mm).
  4. Wash 3x times with 0.1% SDS in PBS for 5 min each.
  5. Resuspend in 1ml 0.1% SDS/ 1% BSA in PBS, Incubate for 30 minutes.
  6. Wash 3x with PBS for 5 min each.
  7. Apply Antibody (1:200 in PBS) for 1 hour in the dark.
  8. Wash 3x in PBS for 5 min each in the dark.
  9. Mount specimen for imaging.
Flow Cytometry - Extracellular Antigens

Protocol 1: Standard protocol

  1. Harvest, wash the cells and adjust cell suspension to a concentration of 1x106 cells in 1ml 1% BSA in PBS, Incubate for 30 minutes.
  2. Spin cells and remove S/N.
  3. Resuspend in 1ml 0.5% BSA. Aliquot 200ul for each test into a new tube (2x105 cells).
  4. Add 1ul Antibody (1:200 final conc.), quick vortex and incubate for 15 minutes in the dark at room temperature.
  5. Wash 3x with 500ul PBS.
  6. Resuspend to a final 500ul PBS and analyse with Flow cytometer.

Protocol 2: Modified protocol with SDS Antigen retrieval step

  1. Harvest, wash the cells and adjust cell suspension to a concentration of 1x106 cells in 1ml 1% BSA in PBS, Incubate for 30 minutes.
  2. Add 1ml 0.02% SDS, spin cells and remove S/N.
  3. Resuspend in 1ml 0.5% BSA. Aliquot 200ul for each test into a new tube (2x105 cells).
  4. Add 1ul Antibody (1:200 final conc.), quick vortex and incubate for 15 minutes in the dark at room temperature.
  5. Wash 3x with 500ul PBS.
  6. Resuspend to a final 500ul PBS and analyse with Flow cytometer.
Flow Cytometry - Intracellular Antigens

Protocol 1: Fix with PFA, Permeabilise with Triton X-100 and SDS (Recommended)

  1. Harvest, wash the cells and adjust cell suspension to a concentration of 1x106 cells per tube.
  2. Spin cells and remove supernatant.
  3. Fix cells by resuspending with 0.5ml PBS and adding 0.5ml 8% Paraformaldehyde to give a final concentration of 4% PFA, incubate for 10 minutes.
  4. Add 5 ml 0.1% SDS in PBS, spin cells and remove S/N.
  5. Wash 1x with 5ml 0.1% SDS in PBS.
  6. Resuspend cells in 1ml Antigen retrieval/Permeabilisation solution (0.1%SDS / 0.1% Triton X-100 / 1% BSA in PBS), Incubate for 30 minutes.
  7. Spin cells and remove S/N, resuspend in 1ml 0.5% BSA in PBS and aliquot 200ul for each test into a new tube (2x105 cells).
  8. Add 1ul Antibody (1:200 final conc.), quick vortex and incubate for 15 minutes in the dark at room temperature.
  9. Wash 3x with 500ul PBS.
  10. Resuspend to a final 500ul PBS and analyse with Flow cytometer.

Protocol 2: Fix with PFA, Permeabilise with Methanol and SDS

  1. Harvest, wash the cells and adjust cell suspension to a concentration of 1x106 cells per tube.
  2. Spin cells and remove supernatant.
  3. Fix cells by resuspending with 0.5ml PBS and adding 0.5ml 8% Paraformaldehyde to give a final concentration of 4% PFA, incubate for 10 minutes.
  4. Add 5 ml PBS, spin cells and remove S/N.
  5. Wash 1x with 5ml PBS.
  6. Resuspend cells in 1ml ice-cold (-20°C) 100% Methanol, incubate for 15 min on ice or 4°C.
  7. Wash 1x with PBS.
  8. Resuspend in 1ml 0.1% SDS/ 1% BSA in PBS, Incubate for 30 minutes.
  9. Spin cells and remove S/N, resuspend in 1ml 0.5% BSA in PBS and aliquot 200ul for each test into a new tube (2x105 cells).
  10. Add 1ul Antibody (1:200 final conc.), quick vortex and incubate for 15 minutes in the dark at room temperature.
  11. Wash 3x with 500ul PBS.
  12. Resuspend to a final 500ul PBS and analyse with Flow cytometer.

Protocol 3: Fix/permeabilise with Methanol, followed by PFA and antigen retrieval with SDS

  1. Harvest, wash the cells and adjust cell suspension to a concentration of 1x106 cells per tube.
  2. Spin cells and remove supernatant.
  3. Fix/Permeabilise cells with 1ml ice-cold (-20°C) 100% Methanol for 15 min on ice or 4°C.
  4. Wash 1x with 5ml PBS.
  5. Resuspend cells with 0.5ml PBS and add 0.5ml 8% Paraformaldehyde to give a final concentration of 4% PFA, incubate for 10 minutes.
  6. Add 5 ml PBS, spin cells and remove S/N.
  7. Wash 1x with PBS.
  8. Resuspend in 1ml 0.1% SDS/ 1% BSA in PBS, Incubate for 30 minutes.
  9. Spin cells and remove S/N, resuspend in 1ml 0.5% BSA in PBS and aliquot 200ul for each test into a new tube (2x105 cells).
  10. Add 1ul Antibody (1:200 final conc.), quick vortex and incubate for 15 minutes in the dark at room temperature.
  11. Wash 3x with 500ul PBS.
  12. Resuspend to a final 500ul PBS and analyse with Flow cytometer.