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Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Unspecific absorption of fluorescein conjugates to tissue has long caused concern. To combat this, several immunofluorescence counterstains have been developed. These also may be helpful for bringing out the background details in colors contrasting with those of specific immunofluorescence and for quenching tissue autofluorescence. One of the best known, Evans blue, was introduced by Nichols and McComb.251 As indicated by its name, it absorbs in the blue region and may therefore also absorb some of the FITC fluorescence.51 When excited at the wavelength normally used for FITC (490 nm), Evans blue gives a deep red fluorescence. In color photomicrographs of strongly counterstained sections, FITC fluorescence appears yellow and not green. The contrast imparted by Evans blue is very nice for color slides, and we have used it very often for demonstration purposes. In everyday laboratory work, use of all types of counterstains should be abandoned, however, due to risks involved in masking weakly antigenic sites. It is far better to prepare good conjugates and good tissue preparations than to deliberately mask unspecific staining. However, for bringing out points already proven, counterstains such as Evans blue certainly amplify the beauty of slide shows! After immunofluorescence staining, sections are dipped in a 0.1% solution of Evans blue in distilled water for 5 to 10 min. Experience is the best guide to this progressively proceeding counterstaining procedure. Additional counterstains have been constructed. Hall and Hansen studied a series of chelated azo dyes and obtained good results with flazo orange.122 This compound is broadly excited with a peak at 535 nm. Its emission peak is in the red region (615 to 630 nm). Like Evans blue, it is therefore useful for counterstaining FITC, but not TRITC. Fey advocated the use of Eriochrome black (diluted 1:60 or 1:30), which brought out a dark red background in immunofluorescence preparations.88 Comparisons among different counterstains were made by Schenk and Churukian, who grouped counterstains into three categories: (1) those eliminating background (Evans blue, Congo red, Flazo orange, and Eriochrome black); (2) those enhancing cellular and tissue detail, and (3) those doing both.303 They argued for the use of a methyl green counterstain for nuclei for defining tissue detail and came out in favor of lissamine-rhodamine-conjugated bovine serum albumin (BSA) and Eriochrome black for reducing unspecific staining and autofluorescence. Evans blue, in agreement with what was stated above, was found to mask some specific fluorescence.303 Interest in blocking unspecific staining of granulocytes (mainly eosinophils) led Johnston and Bienenstock163 to develop (nonfluorescent) staining of granulocytes with phenol chromotrope 2-R stain and Valnes and Brandtzaeg353 to light microscopically stain for endogenous peroxidase before performing immunofluorescence.
Comparison of the effects of operating microscopes with light emitting diode and halogen light source on the eye: a rabbit study
Published in Cutaneous and Ocular Toxicology, 2021
Bahri Aydın, Armagan Ozgur, Huseyin Baran Ozdemir, Pınar Uyar Gocun, Mehmet Arda Inan, Hatice Tuba Atalay, Kemal Bayrakceken, Mehmet Cuneyt Ozmen, Ahmet Yucel Ucgul
Apoptotic cells were searched by TdT-dUTP terminal nick end-labelling (TUNEL) in each half of retinas, and cornea. TUNEL was performed as previously defined using the ApopTag Peroxidase In Situ Apoptosis detection kit (S7110, Millipore, Inc., Temecula, CA, USA), following the manufacturer’s instructions. Peroxidase substrate 3, 3-diaminobenzidine was used to stain for apoptotic cells. Methyl green (0.5%) was used as a nuclear stain. TUNEL positive cells were searched under a light microscope (Olympus, BX51, Japan). The numbers of TUNEL-positive cells in the RGC layer, inner nuclear layer, and outer nuclear layer per retina and cornea were searched in four high power fields (40× objective) The mean cell counts of these sections were used to set the proportion of cells undergoing apoptosis for each layer of each particular eye8.
Topical hyaluronan alone promotes corneal epithelial cell migration whereas combination with benzalkonium chloride impairs epithelial wound healing
Published in Cutaneous and Ocular Toxicology, 2020
Satoshi Seino, Ryosuke Matsuoka, Yasunobu Masuda, Masaaki Kunou, Yuka Okada, Shizuya Saika
To examine the apoptotic effects of HA and BAC on corneal cells, cleaved caspase-3 was analysed by immunohistochemistry34. Briefly, mouse eye samples obtained after in vivo wound healing experiment were subjected to the preparation of formalin-fixed paraffin-embedded tissue sections (thickness: 5 µm). The sections were incubated with anti-cleaved caspase-3 antibodies (1:100; Cell Signalling Technology, Danvers, MA, USA) with blocking serum of VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA) at 4 °C overnight. Then, the slides were incubated with secondary antibodies of the kit (1:200) at 4 °C for 1 h. After further incubation with avidin/biotinylated enzyme complex at 4 °C for 1 h, cleaved caspase-3 was detected by the addition of 3,3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA). The nucleus was counterstained with methyl green.
The intraocular staining potential of anthocyanins and their retinal biocompatibility: a preclinical study
Published in Cutaneous and Ocular Toxicology, 2018
Lan-Hsin Chuang, An-Lun Wu, Nan-Kai Wang, Kuan-Jen Chen, Laura Liu, Yih-Shiou Hwang, Ling Yeung, Wei-Chi Wu, Chi-Chun Lai
For the TUNEL analysis, the eyeballs were harvested after the intravitreal injection and marked for orientation and sagittal sectioning at 5 μm. The tissues were then fixed with 4% paraformaldehyde at 4 °C for 24 h, embedded in paraffin, and sectioned. Sections were dewaxed with xylene and progressively hydrated. Following bisection at the equator to obtain the anterior and posterior segments, the chorioretinal sections from the temporal and nasal quadrants of the posterior segments were assayed. The TUNEL method was performed using an apoptosis terminal deoxynucleotidyl transferase (TdT) DNA fragment detection kit (TdT-FragEL; Oncogene, Darmstadt, Germany), according to the manufacturer’s instructions. Briefly, 5-μm-thick paraffin sections were deparaffinized in xylene and rehydrated using a graded series of alcohol and distilled water. Sections were treated with proteinase K for 15 min at room temperature and washed with distilled water. Endogenous peroxidase was inactivated by incubating the sections with 3% H2O2 for 5 min at room temperature, followed by washes with distilled water. Sections were incubated with biotin-16-dUTP, TdT, and 20% 5X cacodylate buffer in a moist chamber for 1 h at 37 °C and then washed with PBS. Finally, sections were incubated with peroxidase-conjugated streptavidin for 30 min at room temperature and then washed with PBS. Diaminobenzidine was used as a chromogen, and counterstaining was performed with methyl green.