I am analyzing the tissue damage to the mouse cornea tissue after incubated a kind of medium. The protocol is
1) mouse cornea was incubated in the medium for one day
2) embedding the cornea with OCT compound and snap frozen in liquid nitrogen
3) cryosection
4) HE staining
The control is naïve cornea that embeded with OCT, cryosection and HE staining. In fact, the thickness of incubated cornea should be thicker than the naïve one (3-4 times) from the optical coherence tomography. But the thickness of the incubated and naïve cornea from cryosection HE staining is similar. What the problem is? Should I use paraffin section? Why the thickness of incubated cornea is changed after cryosection and HE staining? Many thanks!
use cryosectioning in which the tissue is immersed in cryoprotectant, frozen and then cryosectioned
fresh tissues were immersion-fixed by rapidly replacing the PBS with 10% formalin (Fisher Scientific, Pittsburgh, PA) for 1 minute at room temperature, taken out briefly for imaging and then returned to fixative for 24 hours. To study the effects of sectioning, the 24-hour fixed PPONHs were further processed, including immersing in a 30% sucrose solution for cryoprotection and embedding in Optimal Cutting Temperature compound (Fisher Scientific, Pittsburgh, PA) before freezing in liquid nitrogen (−196 °C). Frozen blocks of PPONH tissue were then cryosectioned coronally at 30 µm thickness (Leica CM3050 S, Leica Biosystems Inc., Buffalo Grove, IL)
use cryosectioning in which the tissue is immersed in cryoprotectant, frozen and then cryosectioned
fresh tissues were immersion-fixed by rapidly replacing the PBS with 10% formalin (Fisher Scientific, Pittsburgh, PA) for 1 minute at room temperature, taken out briefly for imaging and then returned to fixative for 24 hours. To study the effects of sectioning, the 24-hour fixed PPONHs were further processed, including immersing in a 30% sucrose solution for cryoprotection and embedding in Optimal Cutting Temperature compound (Fisher Scientific, Pittsburgh, PA) before freezing in liquid nitrogen (−196 °C). Frozen blocks of PPONH tissue were then cryosectioned coronally at 30 µm thickness (Leica CM3050 S, Leica Biosystems Inc., Buffalo Grove, IL)
Sunny Chi Lik Au Thank you very much for your last suggestion. Due to the lack of some agents, I did it as follows:
1) fresh cornea tissue(has been preincubated for 24 h with a medium and has fully hydrated) in 4%PFA for 20 min
2) wash 3 times with PBS
3) 5% sucrose 30min
4) 30% sucrose 1 hour
5) embedding the tissue in Optimal Cutting Temperature compound and snap-frozen with liquid nitrogen
6) cryosection and HE staining
But, it also can not work. Could you please point out what the problem is? Must I use 10% Formalin instead of 4%PFA? The fixation time is short? How long should I use the sucrose to protect the tissue? Should I also embed the tissue in the O.C.T. compound for a while and how long? Thank you very much!
At what postmortem interval you are taking the cornea? It matters... https://www.researchgate.net/publication/349681978_Postmortem_Ocular_Findings_in_the_Optical_Coherence_Tomography_Era_A_Proof_of_Concept_Study_Based_on_Six_Forensic_Cases