Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Spleen)
Antigen retrieval step
None
Permeabilization
No
Specification
Spleen
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Paraformaldehyde
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% FBS in PBS
Dilution
1/50
Secondary antibody
Dilution
1/200
Name
Non-Abcam antibody was used: Alexa Fluor 633 goat anti-rabbit IgG (H+L)
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 647
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 647
Additional data
Additional Notes
The tissues were taken out of the animal and immediately submerged into a mild fixation buffer containing 1% paraformaldehyde.
The tissues were placed into 30% sucrose for ~ 6 hours before freezing them into OCT tissue compound.
For this experiment, splenic tissue sections of a wt mouse were stained for F4/80 (green) and CD80 (red) and DAPI (blue). CD80 did not show any positive staining.
Abcam response
Thank you for your reveiw of this previously untested sample preparation, PFA-fixed, frozen tissue. We suspect that the antibody is incompatible with PFA or formalin fixation without a subsequent heat-mediated antigen retrieval step. Alternatively, sections of fresh-frozen tissue fixed on the slide with cold acetone or methanol may yield a better result.
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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投稿 Jan 19 2015