Hi everyone
I am working on immunofluorescence of cryosection of murine growth plates. The Ab worked well on 8um cryosection. But when I stained 50um section, I found the fluorescent signals became much weaker after 20um depth (20X lense). I treated my sections with 1% tritonx for 1 hr before blocking, incubated O/N with primary Ab at 4, 2 hr with secondary Ab at RT. How can I reach 100% Ab penetration?
If you aren't already, having the Triton X-100 in your blocking solution and antibody solutions could help.
Check out if it is a penetration problem or detection problem.
If it is penetration problem, then you will see the antibody has penetrated on both the sides of the section upto 20um and the central part will not have any signal. If it is the detection problem then you have to use a multiphoton system or clarify your tissue for imaging. Also, try different section thickness (10, 20, 30, 40, 50 um) to see if it works. Good luck.
@Sathya
If your sections are strong enough, instead of directly adhering them to the slide, float them on detergent-containing buffer, then drain and continue Triton treatment. This will allow permeabilization from both sides and your antibody may penetrate deeper through the section when the section is attached to the slide. You will need to dry the section onto the slides well.
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