Crossed paraformaldehyde

The novolak resins themselves contain no reactive methylol groups and do not form cross-linked structures on heating. If, however, they are mixed with compounds capable of forming methylene bridges, e.g. hexamethylenetetramine or paraformaldehyde, they cross-link on heating to form infusible, thermoset structures. 

Fixation tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 °C and 63 hours at 5°C the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96% followed by 3 x in 99% (V2 hr in each). After additional treatment with 2x2 hrs in petroleum ether (shellsol D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross sections of 12.5 /im were made on a Supercut 2050 Reichart Jung pyramitome. 

Scheme 6.40 Solvent-free crossed Cannizzaro reaction using paraformaldehyde.

The slide preparation is fixed by using either precipitating fixatives such as ethanokacetic acid or cross-linking fixatives such as paraformaldehyde. They are further treated to remove protein with a mixture that includes proteinase K. Removal of proteins facilitates the access of probe to the sample DNA target. 

The so-called crossed Cannizzaro reaction is synthetically more useful than the Cannizzaro reaction itself, as it can be applied for the preparation of alcohols in high yields, without loss of 50% of the product in the formation of the corresponding carboxylic acid. Typically, paraformaldehyde is used as a sacrificial reducing agent, together with the carbonyl compound which is to be transformed into the alcohol. The reaction thus serves as an alternative method to the use of complex hydrides for the reduction of aromatic aldehydes. 

Varma41 was the first to explore the application of microwave irradiation to a crossed Cannizzaro reaction (Scheme 4.21). A mixture of the aldehyde with 2 equiv. of paraformaldehyde and 2 equiv. of Ba(OH)2 8H20 was irradiated in a domestic microwave oven for 0.25-2 min at 900 W. Yields of the alcohols ranged from 80 to 99%, whereas the production of the corresponding carboxylic acid as the by-product could be suppressed to 1-20%. Under thermal conditions in an oil bath at 100-110°C, similar results were obtained although longer reaction times were required. The same reactions attempted with calcium hydroxide failed to provide the Cannizzaro products41. 

Aromatic aldehydes can also be converted selectively into the corresponding benzylic alcohols in a crossed Cannizzaro reaction, if NaOH is used as a base. Similarly, the reaction is performed under solvent-free conditions by mixing the aldehyde with the base and an excess of paraformaldehyde and irradiating with microwave for 20—25 s. An alternative protocol uses 40% formalin solution and basic alumina to obtain comparable yields. The thermal reaction in refluxing methanol was found to require 12 h, providing considerably lower yields of the benzylic alcohols (Scheme 4.22)42. 

To avoid strongly basic conditions that might lead to the formation of by-products with sensitive substrates, a more recent report quotes the use of KF-A12C>3 in combination with paraformaldehyde for the crossed Cannizzaro reaction of aromatic aldehydes. Similarly, microwave irradiation completed the solvent-free reaction within a few minutes, whereas the thermal reaction in dioxane required reaction times of up to 3 h (Scheme 4.23)43. 

The synthetic procedure for these complexes in acetonitrile with paraformaldehyde as a cross-linking agent is described in more detail in Ref 164. 

Cross-linking systems employed are hexamethylenetetramine or paraformaldehyde. The OH function can also be used by reaction with epichlorohydrin to form epoxy novolaks. 

To allow efficient extraction of intact mRNA, it is recommended that alcohol fixatives be used in lieu of cross-linking agents such as paraformaldehyde. In our experience protocols that have been developed for studying paraformaldehyde-fixed paraffin-embedded archival specimens in tandem with RNA amplifications produce low yields of RNA from microdissected brain regions and picked cells. Fixation of tissue with methanol or ethanol preserves immu-noreactivity and mRNA in many cases. Accordingly, we immerse... 

Fixatives Cross-linkers - paraformaldehyde Precipitators -ethanol, methanol. 

Periodate-lysine-paraformaldehyde (PLP) - It is used to increase cross-linking of molecules in the tissue to provide better fixation. Periodate oxidizes sugars attached to lipids and proteins and generates aldehydes, which bind lysine. Paraformaldehyde then cross-links the lysine. It uses 2% paraformaldehyde,... 

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