Abstract
The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,-the challenging integral sectioning tissues, also generated high-quality histological staining sections.
Highlights
12 randomly selected heads were subjected to tissue block staining, followed by tape-aided sectioning in 6 heads and without tape-aided sectioning in 6 remaining heads, and coverslipping with either water-soluble (20% glycerol in water) or oil-soluble mounting medium DPX in 3 heads each
The remaining 12 heads were subjected to traditional histological processing,sectioning with or without adhesive tape aided after tissue block embedding, conventional HE staining of the sections, and mounting
The reproducibility tested on qualified section success rates and slice quality scores with be stated that this technique (BTA) were carried out in four different periods of time
Summary
Recent work in visual neuroscience research has concluded that some degenerative eye diseases follow topographically specific retinal ganglion cell death across the retina [1,2,3,4,5,6,7,8,9,10] and that the anatomical orientation of the retinae with respect to the orbits is important [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32] These studies require visualization of serial whole integrated sections of orbits or eyeballs to determine morphological changes that respond to treatments or genetic modifications. Application of the method in other challenging sectioning tissues (e.g., joints, brains, and so on) produced satisfactory results
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