Monday
May162011

Osmium-free Embedment Protocol 

For a discussion of similar, see:

An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. (1995) Phend KD, Rusioni A, Weinberg RJ. J Histochem Cytochem. Mar;43(3):283-92.   http://jhc.sagepub.com/content/43/3/283

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Fix, cut 50-60 µm sections, rinse well in buffer. All steps on ice packs up to resin. Avoid moisture contamination by condensation.  Times can be varied to suit material and resins.

 

3x5'            0.1M Na acetate (0.1 NaA)

40'            0.1% Tannic Acid in 0.1 NaA (make fresh, keep in dark)

3x5'            0.1 NaA

20'            0.1% CaCl2 + 0.005% Zn acetate in 0.1 NaA (omit Zinc for best immuno) 

2x5'            0.1 NaA

40'            0.1% Uranyl Acetate in 0.1 NaA (keep in dark),

2x5'            0.1 NaA

5'             50%EtOH in 0.05M NaA (50A)(1:1 100% EtOH/ 0.1 NaA)

5'            3pts 50A: 2pts 100%EtOH

5'            80%EtOH

5'            95%EtOH

2x5'            100% EtOH

30'            1:1 100%EtOH/Lowicryl HM-20

30'            1:3 100% EtOH/ Lowicryl HM-20

2x1h             Lowicryl HM-20 (on ice,  at least 2 changes)

Waferize* and UV cure 36-72h over dry ice, in fume hood

*Take a glass slide, place a similarly sized piece of ACLAR plastic on top of it, add some Lowicryl and arrange your sections on it, cover the sections with another piece of ACLAR, and another glass slide. Label as needed, but don’t block the UV light access to the sections.

 

 Lowicryl HM-20 Resin

 

Crosslinker D            2.98g

Monomer E            17.02g

Mix above solutions gently, then add:

Initiator C            0.1g

Mix, hold in foil-wrapped beaker in hood