Application
Immunocytochemistry
Sample
Mouse Cell (RAW264.7 cell line)
Specification
RAW264.7 cell line
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 24°C
Other product details
Dilution
1/200
Incubation time
2 hour(s) and 0 minute(s) · Temperature: 24°C · Diluent: PBS
Secondary antibody
Name
Non-Abcam antibody was used: Vector Goat Anti Rat Biotinylated
Host species: Goat
Clonality: Polyclonal
Conjugation: Biotin
Host species: Goat
Clonality: Polyclonal
Conjugation: Biotin
Dilution
1/200
Additional data
Additional Notes
RAW267.4 cell line was cultured in DMEM/10% FCS on coverslips in 12 well plates.
Cells were washed with PBS and fixed in 4% PFA until staining.
Staining protocol:
Wash 2x2min PBS
Endogenous peroxidase block - 3%H2O2 in methanol for 15mins at RT
Wash 2x2min PBS
Serum block - 10% Goat serum
Primary - tap off serum block add primary in 10% serum for 2h at RT
Wash 2x2min PBS
Secondary - Goat Anti Rat Biotin (Vector) 5ul/ml in 0.1% PBSA 30mins at RT
Wash 2x2min PBS
ABC from Vector 30min at RT
Wash 2x2mins PBS
DAB (Vector) (brown)
Wash 2x2min H2O
Counterstain - Harris' Haematoxylin (purple)
Wash off with H2O
Dehydrate 2mins - 1x75%EtOh 2x100%EtOh 2xHistoclear
Mount using Histomount
Cells were washed with PBS and fixed in 4% PFA until staining.
Staining protocol:
Wash 2x2min PBS
Endogenous peroxidase block - 3%H2O2 in methanol for 15mins at RT
Wash 2x2min PBS
Serum block - 10% Goat serum
Primary - tap off serum block add primary in 10% serum for 2h at RT
Wash 2x2min PBS
Secondary - Goat Anti Rat Biotin (Vector) 5ul/ml in 0.1% PBSA 30mins at RT
Wash 2x2min PBS
ABC from Vector 30min at RT
Wash 2x2mins PBS
DAB (Vector) (brown)
Wash 2x2min H2O
Counterstain - Harris' Haematoxylin (purple)
Wash off with H2O
Dehydrate 2mins - 1x75%EtOh 2x100%EtOh 2xHistoclear
Mount using Histomount
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.
Abcam user community
Verified customer
提交于 Sep 26 2012