HPMA (2-hydroxypropyl methacrylate) is widely used in conjunction with immunocytochemical procedures for its outstanding performance with both soft and hard tissues. Semi-thin HPMA embedded sections for LM and ultra-thin sections for TEM, when stained, yield clearly defined sample areas. HPMA's capacity to preserve tissues and antigens which have been fixed with mild fixative, as well as its penetrability by a variety of enzymes and reagents, make HPMA an outstanding choice for a variety of immunocytochemical procedures. HPMA is less water soluble than GMA, so some dehydration with alcohol is usually needed. Outstanding results from histological specimens is obtained when HPMA is used. Because HPMA does not have to be heated during curing, the specimen's antigenicity is preserved.
HPMA can be cured under cold conditions (0 - 4° C), making it especially useful for immunocytochemical type work in the presence of high lipid content. Or it can be cured at room temperature using benzoyl peroxide.
HPMA monomer and embedding medium are soluble to about 20% volume to volume of water and are miscible with ethanol. Therefore, HPMA infiltrates tissues without the necessity of complete dehydration or the complete removal of a clearing agent.
Both soft and hard tissues, such as bone, cartilage, and teeth ate infiltrated quite readily with HPMA (in comparison to other embedding systems), and is routinely used in conjunction with immuno gold studies agent.
A variety of enzymes, stains and reagents penetrate both semi-thin and ultra-thin sections without the removal of plastic and basic dyes do not stain the low acid plastic to give unwanted background tint.
A "one size fits all" approach does not work so well for HPMA embedding because for TEM applications, one needs thin sections, and therefore a "harder" block is needed, but for LM, one needs thicker sections, and therefore, the need for a "softer" block. Therefore instead of offering a single kit with an intermediate hardness (leaving no one with the optimum hardness), SPI offers two different kits, one specifically optimized for TEM (to yield a harder block) and another one optimized for LM (to yield a softer block).
Furthermore, there are number of advantages to using "low acid" instead of the more common and lower cost "technical grade" HPMA that is generally provided in kits not specifically being labelled as "low acid". For example, at the LM level, the use of "low acid" HPMA (relative to "technical" grade HPMA) results in significantly lower background staining. And at the TEM level, the absence of the acid derivative of the monomer results in less interaction and problems with curing when lipids are present.
For immunogold work, especially in the case of sensitive antigenic sites, the ability to minimize the alcohol dehydration step represents yet another major advantage.
So far as we know, the SPI-Chem Low Acid HPMA kits are compatible will all reported staining methods and procedures.
HPMA embedded tissue samples are stable under the electron beam and can generally be examined without carbon coating.
We are addressing now the cured block, something that to most people is about as inert of a material as one will find. But the standard practice in many laboratories is to use a small jeweler's (or even a small hack) saw to cut the block down to the right size, sometimes even to shape it. We want to address the dust that is generated and how its exposure can and should be minimized.
Storage conditions: Store HPMA under refrigeration, other components can be stored at room temperature.
