Application
IHC - Wholemount
Sample
Mouse Embryo (Wholemount immunostaining of E7.75 mouse embryo)
Specification
Wholemount immunostaining of E7.75 mouse embryo
Other product details
Incubation time
18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBS/0.1% Triton-X/5% donkey serum/1% BSA
Dilution
1/500
Secondary antibody
Dilution
1/500
Name
Non-Abcam antibody was used: Donkey anti rabbit Alexa Fluor® 488
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Additional data
Additional Notes
Immunostaining was observed within the correct cell types at E7.75 and high level of specificity with lot number: GR3203478-2. However, a previous batch/lot number (GR3186553-5) showed high levels of background staining within extraembryonic cell types at both E6.5 and E7.5. Abcam were great at responding to this feedback and recalled and replaced the bad antibody batch with the newer one.
Antibody worked well and comparably at both 1:100 and 1:500 dilution.
Immunostaining protocol:
1. Embryos were fixed for 15 mins at room temperature in 4% PFA.
2. Embryos were permeabilized for 30 mins at room temperature in PBS + 0.5% Triton-X.
3. Embryos were blocked overnight at 4 degrees C in PBST (PBS + 0.1% Triton-X) with 5% donkey serum and 1% BSA.
4. Embryos were incubated overnight at 4 degrees C with primary antibodies diluted in blocking buffer. All Abcam antibodies were tested at 1:100 dilution.
5. Embryos were washed 3x for 15 mins in PBST and blocked for 3 hours in blocking buffer.
6. Embryos were incubated overnight at 4 degrees C with secondary antibodies (Alexa Fluors) diluted 1:500 in blocking buffer.
7. Embryos were washed 3x 15 mins in PBST and stained with a nuclear dye (Hoechst).
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.
DR. Sophie Morgani
Verified customer
投稿 Mar 23 2018