Looking at glycocalyx in kidney tissue samples, wanted to know best method. This is what I have so far:

Day 1 -Collection & Primary Fixation:

1)    Collect tissue and cut the tissue into 1 x 1 mm pieces.

2)    Place 1x1 pieces in a plastic tissue collection dish containing enough 150mM HEPES (pH 7.35) with 30mM MgCl2,1.5% paraformaldehyde, 1.5% glutaraldehyde (fixative) and 20mL 0.5% Alcian Blue 8GX (pH 2.5) at 30 min-RT then 4 degrees overnight.

Day 2 Part A- Postfixation:

3)    Rinse off excess PFA & GA in 150mM HEPES (pH 7.35) with 30mM MgCl2 3 times for 5 minutes.

4)    Place samples in 1% Osmium Tetroxide in 150mM HEPES with 30mM MgCl2 store at 4-degrees for 2-hrs.

5)    Rinse with diH2O of for 5 times for 5 minutes.

6)    En bloc stain in dark with 2% aqueous uranyl acetate for 2-hr at 4-degrees.

Day 2 Part B- Dehydration:

7)    Dehydrate by placing sample in 30% acetone for 15 mins

8)    Place sample in 50% acetone for 15 mins

9)    Place sample in 70% acetone for 15 mins

10) Place sample in 90% acetone for 15 mins

11) Place sample in 100% acetone for 30 mins – repeat this 2 times changing acetone each time.

Day 2 Part C- Resin Embedding (Epon Mix):

12) Without exposing sample to air place sample in Propylene Oxide for 15 minutes repeat again for a further 15 minutes.

13) Place sample in 2:1 mix of Propylene oxide resin for 1-hr

14)Place sample in 1:1 mix of propylene oxide resin for 1-hr

15)Place sample in 1:2 mix of propylene oxide resin for 1-hr

16)Place sample in 100% resin overnight

Day 3 –Resin Embedding Ctd:

17)Place sample in fresh resin in embedding moulds – label sample

 Polymerise at 60-degrees for 12 hours

Any help would be greatly appreciated.