Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (30um-thick brain slices)
Permeabilization
Yes - 0.3% Triton X-100
Specification
30um-thick brain slices
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C
Fixative
Paraformaldehyde
Other product details
Incubation time
48 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 0.3%Triton X-100 + 5%donkey serum in PBS
Dilution
1/200
Secondary antibody
Dilution
1/250
Name
Non-Abcam antibody was used: Alexa Fluor® 594 AffiniPure Donkey Anti-Rabbit IgG
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 594
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 594
Additional data
Additional Notes
IHC-Fr images of TMEM119 (ab209064) and IBA1m monoclonal (Wako Chemicals #016-26721) co-staining in a 30um-thick slice of Wistar rat brain fixed with 4% PFD.
Free-floating sections were blocked with PBS containing 10% donkey serum and 0.3% Triton X-100 for 1 hour at room temperature. Primary antibody incubation (TMEM119 at 1:200, IBA1m at 1:200) for 2 days at 4°C with 5% donkey serum in 0.3% Triton X-100. Samples were then incubated for 4 hours at room temperature with secondary antibodies in PBS containing 5% donkey serum and 0.3% Triton X-100 (donkey anti-rabbit for TMEM119 shown in red and donkey anti-mouse for IBA1 shown in white).
Max projections were generated from Z-stacks taken with a confocal microscope at 1um-intervals at 20x and 60x magnifications.
Some sections were removed after 1-day of primary antibody incubation, washed, incubated with secondary antibodies, and imaged.
The same protocol was also used with TMEM119 alone (no IBA1m) at a 1:500 dilution for 1-day followed by incubation with AlexaFluor 488 donkey anti-rabbit.
In rat cortex, TMEM119 did not stain microglia and hypothalamic tissue showed non-specific signal.
Abcam response
Thank you so much for testing this antibody in rat. We appreciate very much your efforts, but are sorry to hear that the antibody did not work well in this species.
The customer received a discount on a future antibody purchase for testing this product. We welcome additional feedback from other customers as well.
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.
Francesca Mowry
Verified customer
提交于 Jan 04 2018