Hematoxylin staining of paraffin sections.
In plant histology this staining is appropriate for paraffin sections through samples fixed with Navashin. This fixative is a mixture of 1 % chrome trioxide, 37 % formaldehyde and 100% glacial acetic acid in the following proportions 70:28:7 ml.
Steps of the staining protocol.
- Deparaffinize (3 baths of xylol) and hydrate to water using a grade ethanol series, 3 min by step with gently agitation: ethanol 100 2x; ethanol 90; ethanol 70 then distilled water.
- Immerse sections in filtered Harris hematoxylin for 12 to 15 min, according to the kind of specimen and age of the stain.
- Wash in tap water the in distilled water.Mount coverslips with Eukitt or Histokitt
- Optional: 30 sec staining before mounting in a fresh solution of red ruthenium (1:5000) to reveal pectic substances of cell walls.
Principle of hematoxylin stain
This is an example of indirect staining.Hematoxylin is a natural dye extracted from the heartwood of the tropical tree logwood, Haematoxylin campechianum anditself does not stain tissues. It needs to be oxidized to form hematein, the active staining reagent. Oxidation may be obtained by atmospheric oxygen (keep the solutions in an open bottle) or by adding metal alums (the most common is ferric ammonium sulfate) or iodate to the dye solution. The commercial hematoxylin of Harris is ready for use as hematein.
Hematoxylin is a good nuclear stain and heterochromatin as well as chromosomes appear strongly stained. It is also useful for mitosis counting in a section.
Example : staining of longitudinal paraffin sections 8 microns thick through a primary root tip of Zea mays
The two micrographs below show different steps of mitosis in the meristematic region.
Quiescent center (CQ), and the meristem with mitosis (white arrows). mec : calyptrogen.