Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

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.

Definition of a sectioning plane and place for a section containing hoped-for regions using a spare counterpart specimen

“…Laboratory daily work on histology contributes to the development of the technique when we managed to acquire a section that contains a whole intraorbital optic nerve lineament for morphological investigation of the optic nerve and the orbital contents 27 , 28 . Since the optic nerve is widely accepted as a landmark, the sections with the nerve included would be standardized for valid morphometric analysis and comparison.…”

“…The tissue block was balanced in the chamber for at least 45 min before starting sectioning. A serial section of 7 µm in thickness with an interval of 100 µm was conducted as we did before 27 , 28 , 37 , 38 .…”

“…To ensure intact sections were collected at any desired places, the adhesive tape-aided (Adhesive tape, Cat# 57405, Tesa, Germany) sectioning technique was used 28 . Briefly, a slip of the adhesive tape was clipped, held with fine forceps at a corner, and pressed with the adhesive side of the tape onto the trimmed cutting surface.…”

Flow chamber staining modality for real-time inspection of dynamic phenotypes in multiple histological stains

“…For OCT embedding, the trimmed tissue blocks were incubated in OCT for 6 hours, with 3 changes of fresh OCT under sonication at room temperature after incubation in 30% (w/v) sucrose, and snap-frozen in a dry ice/isopentane bath. Consecutive 5 μm thick sections were cut using a Leica microtome—CM1850 cryostat (temperature, − 20°C, Leica Biosystems, Buffalo Grove, IL, USA) and mounted on adhesive slides (Poly lysine slides, Cat#63700-w1, Roth, Germany) for traditional staining and the coated coverslips for flow chamber staining (refer to Fig 2 ), as reports before [ 16 , 36 ]. Five serial sections were cut for heart tissue ( Fig 2A ) and four serial sections for the other tissues ( Fig 2B and 2C ).…”

“…The incubated sections were ready for IF stain. We used the well-established protocol, which has been successfully applied in atherosclerosis and stroke in our laboratory [ 16 , 36 , 37 , 40 – 42 ]. The sections were immunostained for Hu IgG, using an avidin-biotin complex (ABC) method.…”