This is my protocol for rat brain tissue fixation, I doubt it would differ substantially for using ovary tissue (as rodent brain has high fat content)....
1.Tissue must be cut down to appropriate size; no more than 1mm thick in ideally three, but at least in one dimension (for sural nerves and muscles we create pieces of around 1 x 1 x 2-3 mm3 to keep orientation)
2. Primary fixation for at least 3 hours at 4 C in 2% glutaraldehyde in 0.1 M cacodylate buffer (Alternatively, transcardially perfuse animals with Karnovky's fixative).
3. Tissue processing with an automated tissue processor per the below protocol (if manual processing is desired, some of the duplicated or triplicated reagents may be reduced since less carry over of reagent expected) The 2% osmium tetroxide solution for secondary fixation/staining is buffered in 0.1 M cacodylate buffer epon embedding medium is made up as follows: 20 ml Embed-812, 9 ml DDSA, 12 ml NMA, 1.2 ml BDMA (all purchased from Electron Microscopy Sciences/EMS), components are added one by one, with manual stirring in between, final solution is stirred on stir plate for 1 hour before creating mixtures with propylene oxide.
Tissue Processing Solution Steps (Agitate throughout):
0.1M Cacodylate Buffer (5 min at 4 degrees C)
0.1M Cacodylate buffer (5 min at 4 degrees C)
2% Osmium Tetroxide (120 min at 4 degrees C)
0.1M Cacodylate Buffer (5 min at 4 degrees C)
ddH20 (5 min at RT)
50% EtOH (10 minutes at RT)
70% EtOH (10 minutes at RT)
90% EtOH (5 min at RT)
100% EtOH (10 min at RT)
100% EtOH (10 min at RT)
Propylene Oxide (10 min at RT)
Propylene Oxide (10 min at RT)
1:1 Propylene Oxide to Epon (30 min at RT)
1:2 Propylene Oxide to Epon (60 min at RT)
Pure Epon overnight, following by solution change of fresh Epon in the AM. Embed samples in moulds with fresh Epon in the PM.
4. Specimen are embedded in beam capsule or flat molds and cured overnight in an oven at 60 C.
Hope this helps somewhat!
Cut the ovary tissue in small pieces and fix them in karnonovsky for 4-6 hours and after give 0.1 M phosphate buffer changes and stored the tissue in 0.1 M phosphate buffer under 4 degree in freezer until further processing. You can also contact SAIF Faculty in AIIMS for further processing and photography.
- Badges
- Science topic