Overview:

Resin embedding for LM provide greatly improved cellular definition compared to paraffin embedding, and for this reason is now widely used in diagnoses particularly of renal disease, lymphomas and bone marrow trephines, as well as research.

L.R. White can be used for the LM, EM and the histochemical demonstration of some resistant enzymes as well as for the immunohistochemical demonstration of intracellular immunoglobulins.

Fixation:

1.       Fix specimen with 4% paraformaldehyde (or formaldehyde) with or without 2.5% sucrose in 0.1M PB, pH 7.4 for overnight.

2.       Rinse specimen 2x5min with 0.1M PB (or store specimen in 0.2M (8%) sucrose in 0.1M PB for storage and shipping purpose).

Dehydration:

1.       Two changes of 70% ethanol for 30 minutes each.

2.       L.R. White and 70% ethanol (2:1) mixture: slowly add one part of 70% ethanol (drop by drop) to two parts of L.R. White, and shake gently (otherwise the mixture will become milky). Incubate specimen in the mixture for 1 hour.

3.       Incubate specimen in two or three changes of pure L.R. White for 1 hour each, or overnight at room temperature (specimen can be stored in L.R. White at 4 C for weeks if necessary).

Embedding:

1.       Embed specimen in Gelatin Capsules. Place specimen in bottom of the capsule and fill up with L.R. White to the brim and slide the other half of the capsule on.

2.       Polymerize in 60 C oven for 24-48 hours.

Sectioning and Staining:

1.       Cut ultrathin sections and mounted on formvar-carbon coated nickel grids and allow it air-dry overnight.

2.       Perform immunogold labeling procedure (pretreatment such as epitope unmasking with heat may be needed).

3.       Stain with uranyl acetate for 15 minutes and lead citrate for 1-2 minutes.

Observation:

1.       Observe using Transmission Electron Microscopy

2.       Taking pictures