Frozen section

Elimination from the vitreous occurs by one of two pathways. This can be visualized by injecting fluorescent compounds and examining the concentration distribution in frozen sections obtained after a steady state has been established [230]. If the major route of elimination is by means of the re-tina/choroid, at steady state the lowest concentration would be in the vicinity of the retina. The contours observed in frozen sections of the rabbit eye obtained after intravitreal injection of fluorescein exhibit this pattern, with the highest concentration immediately behind the lens (Fig. 16A). Compounds not chiefly eliminated through the retina exit the vitreous by passive diffusion and enter the posterior aqueous, where they are eliminated by the natural production and outflow of aqueous humor. In such a situation, the contours would be perpendicular to the retina, with the highest concentration towards the rear of the vitreous cavity. This appears to be the case for fluorescently labeled dextran polymer, whose contours decrease in concentration toward the hyaloid membrane (Fig. 16B). 

Fig. 16 Contours of fluorescent intensity in frozen sections of the rabbit eye following 15 pL injection of marker solution in the central vitreous cavity injection was conducted through the superior rectus muscle 15 hours following injection of 0.2% sodium fluorescein 14 days following injection of 0.1% FITC-dextran, molecular weight 66,000. (From Ref. 230.)... 

In conclusion, based on the literature and our data, the traditional use of acetone-fixed frozen tissue sections as the gold standard for IHC is not justified for all antigen/antibody pairs. For validating any new antibody, it would be prudent to employ a combination of both acetone and NBF-fixed frozen sections. From our experience, FFPE tissue sections may serve as the standard for most antigens for IHC. 

Mundegar et al.71 applied a modified AR protocol to unfixed frozen sections with the goal of reducing background. Their study was designed to detect a 427 kD subsarcolemmal protein dystrophin in mdx mouse skeletal muscle... 

Yamashita S, Okada Y. Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections. J. Histochem. Cytochem. 2005 53 1421-1432. 

Jylling AMB, Lindebjerg J, Nielsen L, et al. Immunohistochemistry on frozen section of sentinel lymph nodes in breast cancer with improved morphology and blocking of endogenous peroxidase. Appl. Immunohistochem. Mol. Morphol. 2008 16 482-484. 

Frozen sections fixed in acetone have been a favored alternative to formalin-fixed, paraffin-embedded (FFPE) material in critical IHC work, to the point where this was considered the standard against which other fixatives were compared. Recent studies37,38 have now cast doubt on that status, showing that many antigens perform equally well after FFPE (with or without AR) and that nuclear antigens in particular fare better, perhaps because some proteins may be extracted by solvent fixatives. 

Kakimoto K, Takekoshi S, Miyajima K, et al. Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry. J. Mol. Histol. 2008 39 389-399. 

In the case of frozen sections without fixation, a skillful technology is required to stably create slices that are several micrometers thick. For this reason, in most reports, the samples are prepared with slices that are 10-20 pm thick.11... 

Another method is the denaturation of a tissue section with denaturant on the membrane. In this method, the frozen section is thawed and mounted on the membrane. The transferred membrane is washed with 70% ethanol to remove salt and lipid in the tissue and to fix the protein on the membrane. After that, denaturation is processed with the denaturant. Another method is... 

IV. Cutting Sections Ultramicrotomes are designed to cut ultrathin sections, semithin sections and ultrathin frozen sections if suitably... 

Analyses using this approach have revealed more than 1500 protein peaks from histologically selected 1 mm diameter regions of single frozen sections.52 Imaging MS allows investigators to... 

Aldehyde groups stain pink Lipids and fatty acids—Sudan III Unfixed or fixed frozen sections Take sections to 50% aqueous ethanol... 

The future of EM autoradiography most likely will center about the utilization of frozen sections for the localization of soluble compounds within tissues. Of special interest is the expanding application of EM autoradiography to molecular biology. Fakan and Fakan (4) have employed the technique for investigating spread molecular complexes. The reader is referred to Sigre (19) for additional applications of EM autoradiography. 

The ideal solution to microanalysis would be simply to freeze the plant material rapidly to the temperature of liquid nitrogen and then section it while it is still frozen on a cryotome. The frozen sections would then be transferred to a cold stage in a TEM and analyzed. In theory, no ion movement will take place and analysis at the high resolution of TEM should be possible. Indeed, this is a useful technique for liver, kidney, and soft animal tissues, but unfortunately it is almost impossible to cut tough plant material, and maintain the sections in a reasonable state for analysis (2). Even if this problem could be overcome unstained tissues will be difficult to visualize in TEM. 

Yamashita S. Intranuclear localization of hormone-occupied and -unoccupied estrogen receptors in the mouse uterus application 1 nm immunogold-silver enhancement procedure to ultrathin frozen sections. JElectron Microsc 1995 44 22-29. 

C. These freeze-dried sections were dry mounted on microscope slides which had been precoated with either Kodak NTB-3 or NTB-10 emulsion. Other techniques which thawed the frozen section, embedded the tissue in paraffin or dipped the section in liquid emulsion were demonstrated to translocate diffusible compounds. Many other similar attempts have been and are currently being made to localize diffusible compounds by autoradiography at the electron microscope level. 

Cut frozen sections at 5 10 pm. Pick up on adhesive-coated slides. 

Cut frozen sections with a cryostat and mount them on glass slides coated with freshly prepared 0.02% poly-L-lysine. Free-floating sections can also be used. 

Antigenic determinants masked by formalin-fixation and paraffin-embedding may also be exposed by enzymatic digestion. This can, however, not be used with frozen sections or cells which are not paraffin-embedded. The beneficial effects of protease treatment are presumably related to cleavage of the molecular cross-links by the... 

Stain embedded sections lightly in uranyl acetate and lead citrate (optional). Frozen sections may be stained with osmium tetroxide vapor. Staining with OSO4 or uranyl acetate may be conducted without obscuring the gold particles. Wash and examine under the electron microscope. 

Stumpf WE, Roth LJ. 1966. High resolution autoradiography with dry mounted, freeze-dried frozen sections. Comparative study of six methods using two diffusible compounds H-estradiol and H-mesobilirubinogen. J Histochem Cytochem 14 274-287. 

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