Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa cell line)
Permeabilization
No
Specification
HeLa cell line
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 25°C
Fixative
Methanol
Other product details
Incubation time
30 minute(s) · Temperature: 25°C · Diluent: 2% BSA PBS
Dilution
1/300
Secondary antibody
Dilution
1/100
Name
Non-Abcam antibody was used: RITC secondary antibody
Host species: Goat
Clonality: Monoclonal
Conjugation: Rhodamine
Host species: Goat
Clonality: Monoclonal
Conjugation: Rhodamine
Additional data
Additional Notes
The methanol fixation time was 10 min (on ice).
Immunostaining of formaldehyde-fixed cells was also performed in a similar manner to that described above but it requires the permeabilization step. It was carried out using NetGel plus 0.25% Nonidet P-40 for 10 min on a shaker at RT.
NETgel composition: [150 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl pH 7.4, 0.05% Nonidet P-40 (NP-40, Roche, 11754599001), 0.25% gelatin and 0.02% Na azide]
Cells were permeabilized for 10 min on a shaker at room temperature. Following permeabilization, samples were washed twice in NETgel to remove the extra detergent before staining. NETgel was then used for all antibody dilutions and washes.
The immunostaining was good with both methods of fixation.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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提交于 Jun 18 2015