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Intracellular Double Labeling of Substantia Nigra and Pedunculopontine Neurons in in vitro Slice Preparation
Published in Avital Schurr, Benjamin M. Rigor, BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
In order to first identify biocytin-injected neurons, sections are incubated in Texas Red avidin (1:250 to 1:500 dilution, Vector) with 0.1 M PBS containing 0.3% Triton™ X-100 for 2 h at room temperature. Plastic tissue culture plates with lids are convenient vessels for carrying out the immunocytochemical procedures. Second, the transmitter phenotype of the recorded cell is identified by the following immunocytochemical procedures. First, the sections are rinsed in 0.1 M PBS three times (10 min each). This thorough rinsing is necessary to remove any unconjugated Texas Red avidin. Sections are incubated in primary antibody (e.g., TH) with 0.1 M PBS containing 0.3% Triton™ X-100 and 3% normal serum obtained from the same host species as the secondary antibody (e.g., normal goat serum if the secondary antibody to the rabbit IgG [primary antibody] is from goat) for 24 to 48 h at 4°C. One should carefully follow the suggested dilution and incubation time specified with the product, since these factors are specific for each antibody. Sections are again rinsed three times (10 min each) in 0.1 M PBS. Next, sections are incubated in fluorescein isothiocyanate (FITC) conjugated secondary antibody (1:150 dilution, Vector) with 0.1 M PBS containing 0.3% Triton™ X-100 for 3 h at room temperature. Following three 10-min rinses, sections are mounted on gelatin-coated glass slides and coverslipped with PBS containing 50% glycerol and 2.5% DABCO (1,4-diazabicyclo [2.2.2] octane), which retards photobleaching of fluorescent material during microscopy. Sections are examined under an epifluorescence microscope using the appropriate filter system for Texas Red and FITC.
Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Several attempts have been made to combat the fading phenomenon, particularly with FITC. Normally, sections are mounted in a mixture of glycerin and phosphate or Tris buffer, pH 7.0 to 7.4. Fading is somewhat retarded by mounting at higher pH (8.6) or by mounting in nonfluorescent, xylene-miscible media such as Entellan.®81,243 Lennette suggested use of polyvinyl alcohol (PVA), buffered to pH 9, to store FITC-stained specimens.190 Reduction of the fading of FITC and rhodamine was obtained with (1) n-propyl gallate,107 (2) 1 mg/mℓ paraphenylenediamine (PPD) in glycerin,161 (3) 1 mg/mℓ 1,4-diazobicyclo- (2,2,2)-octane (DABCO) in gelvatol,183 or (4) 25 mg/mℓ DABCO in phosphate-buffered glycerin.162 The advantage with DABCO was the finding that, in contrast to other mounting media, its addition did not destroy the specimens upon storage.183 A comparison was more recently made by Valnes and Brandtzaeg, who recommended use of polyvinyl alcohol medium containing 0.2 to 2 g/ℓ of PPD or 6 g/ℓ of n-propyl gallate.354 These authors found no advantage with DABCO, except that it permitted long-term storage. There are several considerations in the use of these mounting media (effects on TRITC contra FITC, effects on section durability, effects on tissue background fluorescence). We have never used them, but have relied upon inspection of freshly stained sections mounted in glycerine:buffer (9:1). The interested reader should consult the original publications before embarking on this avenue for prolonging fluorescent life. Conceivably, the methods could be extremely helpful for preventing fading of weakly fluorescent structures during photography. Upon storage of conventionally mounted immunofluorescence preparations, fluorescence is gradually lost. Weinstein and Lechago observed that fluorescence in indirect immunofluorescence-stained preparations (as well as in sections poststained with hematoxylin-eosin) could be regained by renewed application of the FITC-labeled second antibody.374 Conceivably, this could indicate loss of fluorescence due to dislodgement of the fluorochromed second antibodies during storage, but this question was never addressed. Nevertheless, we have repeatedly used the Weinstein and Lechago approach also on semithin plastic sections that have faded in the fluorescence microscope (Larsson, unpublished data) and have found it to work very well.
Synthesis of N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles enabling PKBβ/AKT2 inhibitory and in vitro anti-glioma activity
Published in Annals of Medicine, 2022
Ruturajsinh M. Vala, Vasudha Tandon, Lynden G. Nicely, Luxia Guo, Yanlong Gu, Sourav Banerjee, Hitendra M. Patel
In the current work, we used DABCO as a catalyst to synthesise N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles. DABCO is a highly reactive, inexpensive, eco-friendly, nontoxic catalyst for various organic transformations [32]. As shown in Scheme 2, two types of DABCO catalysed four-component synthesis of pyrano[2,3-c]pyrazoles are available. The first method is similar to Scheme 1(c). In the second method, the aldehyde is replaced by dimethyl acetylenedicarboxylate. Both methods produce pyrano[2,3-c]pyrazoles, but substitutions at C-4 are different. In continuation of works on aldehyde based three-component reactions [33–39], in current work, we synthesised N-(4-chlorophenyl) substituted pyrano[2,3-c]pyrazoles by aldehyde based DABCO catalysed three-component reaction (Scheme 3). We further report 4j as the first pyrano[2,3-c]pyrazole derivative with kinase inhibitory and anti-glioma potency from within the series. Compound 4j inhibited AKT2/PKBβ specifically among 139 purified kinases tested and induced cell death in primary patient-derived glioma 2D cells and 3D neurospheres while being relatively nontoxic towards non-cancerous cells.
The piperazine scaffold for novel drug discovery efforts: the evidence to date
Published in Expert Opinion on Drug Discovery, 2022
Maria Novella Romanelli, Dina Manetti, Laura Braconi, Silvia Dei, Alessio Gabellini, Elisabetta Teodori
Maralixibat (15, Table 1) is an ileal bile acid transporter (IBAT) inhibitor approved for the treatment of rare cholestatic liver diseases, including Alagille syndrome [50]. This compound was originally described in a paper on the development of IBAT inhibitors as an approach to decrease serum LDL cholesterol [51]. The piperazine ring in this compound is part of a 1,4-diazabicyclo[2.2.2]octane (DABCO) moiety. One N atom has been quaternarized in order to avoid systemic exposure: in fact, IBAT is localized on the luminal surface of the distal ileum and the inhibitor must bind within the intestine. Among all the cationic groups that have been tested, the DABCO moiety gave a potent, crystalline and non-hygroscopic compound; these favorable properties could be also due to the insertion of an aromatic moiety, as rigid backbone, able to provide intermolecular π-π stacking interactions to stabilize the crystal lattice.
Dstac is required for normal circadian activity rhythms in Drosophila
Published in Chronobiology International, 2018
I-Uen Hsu, Jeremy W. Linsley, Jade E. Varineau, Orie T. Shafer, John Y. Kuwada
Adult Dstac-gfp trap brains were dissected in ice-cold 1xPBS and fixed immediately in 4% paraformaldehyde in 1xPBS for 30 minutes at RT. The brains were rinsed with 1xPBSTX-100 for 5 times, 15 minutes/time. The brains were blocked with 10% NGS in 1xPBS with 2% TX-100 for 2 h at RT. The brains were incubated with mouse anti-PDF (Blau 2005)(1:500, PDF C7 (deposited by Blau, Justin (DSHB Hybridoma Product PDF C7)) in 1xPBS with 1% NGS and 0.25% TX-100 (dilution buffer) overnight at RT. The brains were washed with 1xPBS with 3% NaCl and 1% TX-100 (wash buffer) for 3 times, 15 minutes/time. We incubated the brains with anti-mouse Alexa Fluor 647 (1:1000, Invitrogen) in dilution buffer overnight at RT. The brains were then washed with wash buffer and incubated with chicken anti-GFP (1:5000, Aves Laboratories) and anti-chicken Alexa Fluor 488 (1:2000 Jackson ImmunoResearch) following the same procedures described above. The brains were mounted with DABCO FluoroGel.