THIOCARBOHYDRAZIDE-SILVER PROTEINATE STAINING (TCH+SP) AND PERIODIC ACID-THIOCARBOHYDRAZIDE-SILVER PROTEINATE STAINING (PA+TCH+SP)
The endexine is a frequently misinterpreted layer of the pollen wall. Using standard TEM staining techniques (uranyl acetate and lead citrate), ektexine and endexine may differ in their electron opaqueness in that the endexine is higher in electron density than the ektexine, or the situation may be reversed . But in many species, especially when the endexine is thin and less compact or discontinuous, the differentiation of the two layers is insufficient.
The endexine can be differentiated from the ektexine and the intine by thio-carbohydrazide-silver proteinate (TCH+SP) staining in osmium-fixed material and periodic acid-thiocarbohydrazide-silver proteinate (PA+TCH+SP) staining in osmium-free material. The endexine stains electron dense after TCH+SP staining , indicating lipidic compounds, and electron translucent after PA+TCH+SP staining , excluding this layer as part of the intine, as it is well known that intine reacts positively for polysaccharides.
A morphological characteristic of the endexine is its increasing thickness close to the aperture.
PA+TCH+SP staining (localization of neutral polysaccharides):
Sections (80-100 nm) from osmium-free material are placed on gold grids and treated with 1 % PA for 45 min, 0.2% TCH for 8-15 h, and 1 % SP for 30 min (THIERY 1967).
TCH+SP-staining (detection of unsaturated lipids): Sections (80-100nm) from osmified material are placed on gold grids and treated with 0.2 % TCH for 8-15 h and 1 % SP for 30 min (ROWLEY and DAHL 1977, WEBER 1992).
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