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Fixation and Tissue Pretreatment
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Cryostat sections, carefully frozen after cryoprotection, have also been quite extensively used for pre-embedding immunocytochemistry.17,30,54,63,72,90,101,128 Fixations employed have been simple formaldehyde fixation, the PLP fixative, or glutaraldehyde either alone or combined with formaldehyde.17,30,54,63,72 Useful ultrastructural preservation with higher formaldehyde concentrations, formaldehyde-glutaraldehyde combinations, glutaraldehyde, and the PLP fixative has generally been obtained. Using the 3% paraformaldehyde: 2% glutaraldehyde fixative described in the previous section, we have obtained excellent ultrastructural preservation. All of the studies referred to have employed peroxidase-based methods for pre-embedding immunocytochemistry. Some methodological details are given in the Appendix. A comparison between pre- and postembedding methods for localization of gonadotropic hormones in pig pituitary was published by Dacheux.27 This author found the pre-embedding method to permit visualization of hormone both in secretory granules, Golgi complex, and rough endoplasmic reticulum of the gonadotropic cells. Postembedding staining, in contrast, permitted only staining of secretory granules. Dacheux interprets this to mean that embedding in Epon® makes antigens in the Golgi and rough endoplasmic reticulum inaccessible for reaction.27
Glycogenosis type III/amylo-1, 6-glucosidase (debrancher) deficiency
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop
Histologic examination reveals the cells of the liver to be swollen and finely granular with an open nucleus. The material stored may be identified by Best stain as glycogen. Large vacuoles in the hepatocytes may be filled with periodic acid Schiff (PAS)-positive material. There is evidence of an increased amount of fibrous tissue within the lobules of the liver. In addition, there may be some proliferation of the bile ducts. Unlike the picture in glycogenosis type I, there is no infiltration with fat. Histologic examination of the muscle reveals abundant amounts of glycogen visible in subsarcolemmic areas of myofibrils [17]. The glycogen in this disease is more soluble than a normal glycogen and, therefore, it tends to disappear more readily from conventional histologic preparations. Cryostat sections may be useful for biopsies.
Autoradiography
Published in Howard J. Glenn, Lelio G. Colombetti, Biologic Applications of Radiotracers, 2019
Sven Ullberg, Bengt Larsson, Hans Tjälve
Several different types of cryostat equipment have been constructed, and a few of them have been marketed. Two advanced models which were relatively recently developed will be described shortly. They differ mainly in size. The large one (LKB® 2250) allows the sectioning of such large specimens as a 5 kg pregnant monkey 45 cm × 15 cm (Figure 4). The other one can be used for mice and rats 16 cm × 15 cm (LKB® 2258). Both can also be used for small specimens such as rat fetuses. The good stability also allows the sectioning of such hard tissues as teeth. The sectioning is done on a specially designed heavy-duty microtome, which is built into the cryostat. The motor driving the microtome sledge is maneuvered automatically or by a knee control unit with adjustable speed. The knife-holder arrangement is very stable; the pillars holding the knife move vertically. The thickness of the section is determined by the downward feed of the knife, the specimen holder being moved only horizontally. The section thickness can be varied in a continuous range from 1 to 999 μm.
Clinicopathological characteristics and predictors of poor outcome in anti-glomerular basement membrane disease – a fifteen year single center experience
Published in Renal Failure, 2021
Zafirah Zahir, Asif Sadiq Wani, Narayan Prasad, Manoj Jain
Renal biopsy evaluation: Two core biopsies, taken under ultrasound guidance by biopsy gun, one for light microscopy and immunofluorescence each, were evaluated by either of the two nephropathologists. For light microscopy, standard sections were cut from renal biopsy received in 10% buffered formalin. Biopsies were evaluated for endocapillary, extracapillary or mesangial proliferation, fibrinoid necrosis, interstitial inflammation, percentage of glomerulosclerosis, interstitial fibrosis, and tubular atrophy. For imunofluorescence, the biopsy was transported in normal saline. Standard sections were cut in cryostat at a temperature of −20 °C. The percentage of sclerosed glomeruli were graded as Grade 1(<25%), Grade 2 (25–50%), and Grade 3 (>50%). Interstitial fibrosis and tubular atrophy were graded as mild (<25%), moderate (25–50%), and severe (>50%). Interstitial inflammation was also graded as mild (<25%), moderate (25–50%), and severe (>50%).
Mass spectrometry-based phospholipid imaging: methods and findings
Published in Expert Review of Proteomics, 2020
Al Mamun, Ariful Islam, Fumihiro Eto, Tomohito Sato, Tomoaki Kahyo, Mitsutoshi Setou
– Before slicing, frozen tissues are transferred into a cryostat chamber and left for 15–20 minutes at – 10°C to – 20°C to equilibrate to the sectioning temperature. Tissue block is affixed to the stage of a cryostat machine using an embedding media and then sliced at a thickness of around 5–20 µm. For 3D MSI, a series of serial sections are collected with minimal error and registered carefully. During sectioning, the optimized temperature of the cryostat chamber must be maintained depending on the type of the tissue. Particular care should be taken when using optimal cutting temperature embedding media (OCT) or other embedding media containing polymer as they cause serious contamination reducing the quality of the spectra. The thickness of the section should be as thin as possible to achieve higher quality spectral data in MALDI-MSI [49], although DESI-MSI has the flexibility regarding the sample thickness.
Bupivacaine Injection in the Extra Ocular Muscle of Rabbits: Analysis of Global and Orbital Layers
Published in Current Eye Research, 2020
Luisa Moreira Hopker, Marco Aurélio Senff de Moraes, Rodrigo Nitsch, Gabriela Wahab Pasqual, Pamela Cavagnari, Solena Ziemer Kusma, Luciane Moreira, Edmar Zanoteli, Norma Allemann
The muscles were excised and embedded in Tissue-Tek, frozen in isopentane cooled in liquid nitrogen and stored at −80°C until processing. The transverse sections (8–10 µm) were cut on a cryostat at a temperature of −25°C. All the sections analysed were cut 6 mm from the insertion, that is, near the site of BUP application. The immunoperoxidase technique was used to analyse the myosin subtypes. The endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 min. After washing the slides thrice in PBS, the sections were blocked with 1% bovine serum albumin, 0.5% Tween-20 and 10% normal goat serum in PBS and incubated overnight at 4°C with a primary antibody diluted in a blocking solution. The sections were rinsed in PBS, incubated with biotinylated anti-rat secondary antibodies for 1 h (only for reticulin), rinsed in PBS and then incubated with ABC reagent (Vector Laboratories) for 1 h. The slides were rinsed in PBS and incubated with 3,3′-diaminobenzidine substrate solution for 2 min. The antibody used was anti-slow myosin (mouse clone A4.951, Hybridoma Bank).