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Abstract Ethylenediaminetetraacetic acid (EDTA), a classically used chelating agent of decalcification, maintains good morphological details, but its slow decalcification limits its wider applications. Many procedures have been reported to accelerate EDTA-based decalcification, involving temperature, concentration, sonication, agitation, vacuum, microwave, or combination. However, these procedures, concentrating on purely tissue-outside physical factors to increase the chemical diffusion, do not enable EDTA to exert its full capacity due to tissue intrinsic chemical resistances around the diffusion passage. The resistances, such as tissue inner lipids and electric charges, impede the penetration of EDTA. We hypothesized that delipidation and shielding electric charges would accelerate EDTA-based penetration and the subsequent decalcification. The hypothesis was verified by the observation of speedy penetration of EDTA with additives of detergents and hypertonic saline, testing on tissue-mimicking gels of collagen and adult mouse bones. Using a 26% EDTA mixture with the additives at 45°C, a conventional 7-day decalcification of adult mouse ankle joints could be completed within 24 h while the tissue morphological structure, antigenicity, enzymes, and DNA were well preserved, and mRNA better retained compared to using 15% EDTA at room temperature. The addition of hypertonic saline and detergents to EDTA decalcification is a simple, rapid, and inexpensive method that doesn't disrupt the current histological workflow. This method is equally or even more effective than the currently most used decalcification methods in preserving the morphological details of tissues. It can be highly beneficial for the related community.

Abstract Histological examination of targets in regions of interest in histological sections is one of the most frequently used tools in biomedical research. However, it is a technical challenge to secure a multitarget section for inspection of the structure’s mutual relationship of targets or a longitudinally filamentous- or tubular-formed tissue section for visitation of the overall morphological features. We present a method with a specified cutting plane and place, allowing researchers to cut directly at the multitarget centers accurately and quickly. The method is proven to be reliable with high accuracy and reproducibility and a low coefficient of variation, testing on repeat experiments of three target’s position-known models. With this method, we successfully yielded single sections containing whole intraorbital optical nerves, three aortic valves, or whole thoracic tracheas in their central positions. The adjoined custom-made tools used in the study, such as various tissue-specific formulated calibrated trimming and embedding guides, an organ-shaped cavity plaster mold, and a two-time embedding technique for optimal and identical trimming or embedding, also bear great potential to become a common supplemental tool for traditional histology and may contribute to the reduction of the labor, and the number of animals needed.

Traditional histological stains, such as hematoxylin-eosin (HE), special stains, and immunofluorescence (IF), have defined myriads of cellular phenotypes and tissue structures in a separate stained section. However, the precise connection of information conveyed by the various stains in the same section, which may be important for diagnosis, is absent. Here, we present a new staining modality-Flow chamber stain, which complies with the current staining workflow but possesses newly additional features non-seen in conventional stains, allowing for (1) quickly switching staining modes between destain and restain for multiplex staining in one single section from routinely histological preparation, (2) real-time inspecting and digitally capturing each specific stained phenotype, and (3) efficiently synthesizing graphs containing the tissue multiple-stained components at site-specific regions. Comparisons of its stains with those by the conventional staining fashions using the microscopic images of mouse tissues (lung, heart, liver, kidney, esophagus, and brain), involving stains of HE, Periodic acid-Schiff, Sirius red, and IF for Human IgG, and mouse CD45, hemoglobin, and CD31, showed no major discordance. Repetitive experiments testing on targeted areas of stained sections confirmed the method is reliable with accuracy and high reproducibility. Using the technique, the targets of IF were easily localized and seen structurally in HE- or special-stained sections, and the unknown or suspected components or structures in HE-stained sections were further determined in histological special stains or IF. By the technique, staining processing was videoed and made a backup for off-site pathologists, which facilitates tele-consultation or -education in current digital pathology. Mistakes, which might occur during the staining process, can be immediately found and amended accordingly. With the technique, a single section can provide much more information than the traditional stained counterpart. The staining mode bears great potential to become a common supplementary tool for traditional histopathology.

Abstract Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease which affects primarily the joints. Peptides of several proteins have shown an effect in some experimental animal models of RA. We investigated arthritis development in male DBA/1 mice which were injected with bovine collagen II (bCII) and human fibrinogen (hFib) on days 0 and 21, leading to stable and reproducible disease induction in 100% of immunized mice (FIA-CIA). In a second study, two bCII—derived peptides were given three times in the course of 6 weeks after FIA-CIA induction to test for impact on arthritis. Mice were scored weekly for arthritis and anti-citrullinated peptide antibodies (ACPAs) were determined in the sera taken on days 0, 14, 35, 56 and 84. Histology of the hind paws was performed at the end of the experiment. Intravenous administration of peptide 90578, a novel fructosylated peptide derived from the immunodominant T cell epitope of bCII, at a dosage of 1 mg/kg resulted in significant beneficial effects on clinical outcome parameters and on the arthritis histology scores which was sustained over 12 weeks. Survival tended to be improved in peptide 90578-treated mice. Intravenous administration of pure soluble peptide 90578 without adjuvants is a promising approach to treat RA, with treatment starting at a time when ACPAs are already present. The results complement existing data on peptide “vaccination” of healthy animals, or on treatment using recombinant peptide expressing virus or complex biological compounds.

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.