A21071

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

The A21071 is a lab equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a core function within a laboratory setting. However, due to the need to maintain an unbiased and factual approach, a detailed description of the product's intended use is not available at this time.

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12 protocols using a21071

Immunostaining of Mouse Brain Sections

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For immunostaining, mice were deeply anesthetized with Fatal‐Plus and perfused with cold PBS, followed by 4% paraformaldehyde in PBS, pH 7.4. Brains were removed and post‐fixed overnight at 4°C and submerged in 30% sucrose. Forty‐micrometer‐thick sections were cut on a sliding microtome, blocked in 5% normal goat serum, and incubated overnight at 4°C in biotin‐conjugated WFA (1:1000; Sigma‐Aldrich L1516) to label PNNs, rabbit anti‐PCP4 (1:500, SCBT, sc‐74186) or mouse anti‐regulator of G‐protein signaling 14 (RGS‐14) (1:500, UC Davis/NIH NeuroMab Facility, AB_10698026) to label CA2 pyramidal cells, or guinea pig anti‐ZNT3 antibody (1:500, Synaptic systems #197 004) to label axons from the dentate gyrus (mossy fibers). Sections were washed three times in PBS and incubated in combinations of the following secondary antibodies at 1:500 for 40 min at room temperature: streptavidin Alexa‐488 (Invitrogen #S11226), goat anti‐rabbit A633 (Invitrogen #A21071), goat anti‐mouse A488 (Invitrogen # A11008), or goat anti‐guinea pig A633 (Invitrogen #A21105). Sections were mounted with Vectashield antifade mounting medium with DAPI (Vector Laboratories #H‐1500). Images were acquired on a Zeiss laser scanning confocal (LSM510 NLO) or a Zeiss light microscope using controlled camera settings. Images from the different wavelengths were pseudo‐colored for figure clarity.

Carstens K.E., Gloss B.R., Alexander G.M, & Dudek S.M. (2020). Modified adeno‐associated virus targets the bacterial enzyme chondroitinase ABC to select mouse neuronal populations in vivo using the Cre‐LoxP system. The European Journal of Neuroscience, 53(12), 4005-4015.

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Tissue Preparation and Immunostaining of Mouse Brain Sections

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Mice were perfused trans-cardially with PBS followed by 4% paraformaldehyde fixation solution. Brains were dissected and post-fixed in fixation solution at 4°C overnight, soaked in 30% sucrose solution overnight, embedded in O.C.T. (Tissue-Tek, Sakura Finetek USA, INC., Torrance, CA), frozen, and cut into 20–35 μm coronal sections. After washing, sections were blocked for 1 hr (3% normal goat serum in PBS, 0.4% Triton X-100, 0.2% sodium azide) followed by incubation with primary antibody: chicken anti GFP (abcam, ab13970), rabbit anti-Adcy3 (Santa Cruz Biotechnology, sc-588) or mouse anti FLAG M1 (Sigma, F3040) overnight at 4°C. Sections were extensively washed in PBS, and then incubated with secondary antibody: goat anti-chicken Alexa fluor 488 (Invitrogen, A11039), goat anti-mouse Alexa fluor 488 (Invitrogen, A11001), or goat anti-rabbit Alexa fluor 633 (Invitrogen, A21071).

Siljee J.E., Wang Y., Bernard A.A., Ersoy B.A., Zhang S., Marley A., Von Zastrow M., Reiter J.F, & Vaisse C. (2018). Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity. Nature genetics, 50(2), 180-185.

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Immunostaining of Retinal Cell Markers

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One retina from each mouse was fixed, cryo-sectioned and immunostained as described before^{9 (link)}. The cryosections were immunostained with rabbit anti- PKCα (1:10,000), guinea pig anti-GP (1:2000), mouse anti-Brn3A (1:150) or mouse anti-Rho (1:1000) and fluorescently labeled (Alexa Fluor goat anti-guinea pig 488; 2μg/ml; Invitrogen, A-11073; Alexa Fluor goat anti-mouse 555; 2μg/ml; Invitrogen, A-21424; Alexa Fluor goat anti-rabbit 633; 2μg/ml; Invitrogen, A-21071 and nuclei were stained histologically with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; MilliporeSigma) as described before^{9 (link)}.

Grillo S.L., Montgomery C.L., Johnson H.M, & Koulen P. (2018). Quantification of changes in visual function during disease development in a mouse model of pigmentary glaucoma. Journal of glaucoma, 27(9), 828-841.

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Comprehensive Immunofluorescence Imaging Protocol

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Cells were fixed in 4% PFA for 10 min, permeabilized in 0.5% Triton X-100/phosphate-buffered saline (PBS) for 20 min, and then blocked in 5% bovine serum albumin/PBS for 30 min. Fixed cells were stained with the following primary antibodies: TRI-1–60 (1:200, ab16288; Abcam), OCT4 (1:200, ab18976; Abcam), SOX2 (1:200, ab92494; Abcam), cTnI (1:200, ab92408; Abcam), cTnT (1:200, ab8295; Abcam), and α-actinin (1:200, A5044; Sigma). These primary antibodies were visualized with AlexaFluor 488 (1:1000, A11029; Invitrogen) or AlexaFluor 633 (1:1000, A21071; Invitrogen). TUNEL staining was performed with a commercial kit according to manufactory menu (T18120; Yeasen Biotech). GFP-SORBS2 plasmids were transfected into HEK293 cells. After 48 hr, cells were fixed with 4% PFA for 20 min and treated with 0.1% triton X-100 for 10 min. F-actin was stained with Acti-stainTM 555 Fluorescent Phalloidin (Cat. #PHDH1; Cytoskeleton). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescent images were acquired using a Laser confocal microscope (Leica TCS SP8).

Liang F., Wang B., Geng J., You G., Fa J., Zhang M., Sun H., Chen H., Fu Q., Zhang X, & Zhang Z. (2021). SORBS2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome patients. eLife, 10, e67481.

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Immunofluorescence Analysis of Autophagy

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2 × 10⁴ T84 cells were seeded on glass coverslips and grown to 20% confluency and cells were processed as for western blotting experiments. After the indicated infection time, cells were washed 3 times with PBS, fixed with 3% paraformaldehyde, blocked with PBS containing 3% bovine serum albumin (Sigma Aldrich, A7030), and incubated with specific antibodies as indicated in the analysis of autophagy section. Secondary anti-mouse Alexa-488 and secondary anti-rabbit Alexa-633 conjugated antibodies were used (Invitrogen, A21202 and A21071 respectively. Coverslips were mounted in Prolong Gold antifade reagent (Molecular probes, P36935) with the nuclei-staining dye DAPI (Life Technologies, P36935) and visualized with a Zeiss Axiophot confocal fluorescence microscope (Marly le Roy, France). For quantification, 30 to 50 GFP-LF82 bacteria, on 3 different images, were counted and assayed for their colocalization with LC3-II, LAMP1, EEA1, ATG16L1 and ULK1. Each experiment was repeated at least 2 times.

Mimouna S., Bazin M., Mograbi B., Darfeuille-Michaud A., Brest P., Hofman P, & Vouret-Craviari V. (2015). HIF1A regulates xenophagic degradation of adherent and invasive Escherichia coli (AIEC). Autophagy, 10(12), 2333-2345.

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Tissue Preparation and Immunostaining of Mouse Brain Sections

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Immunohistochemical Labeling of Mouse Brain

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For immunostaining, mice were deeply anesthetized with Fatal-Plus and perfused with cold PBS, followed by 4% paraformaldehyde in PBS, pH 7.4. Brains were removed and post-fixed overnight at 4°C and submerged in 30% sucrose. Forty-micrometer-thick sections were cut on a sliding microtome, blocked in 5% normal goat serum and incubated overnight at 4°C in biotin-conjugated WFA (1:1000; Sigma-Aldrich L1516) to label PNNs, rabbit anti-PCP4 (1:500, SCBT, sc-74186) or mouse anti-regulator of G-protein signaling 14 (RGS-14) (1:500, UC Davis/NIH NeuroMab Facility, AB_10698026) to label CA2 pyramidal cells, or guinea pig anti-ZNT3 antibody (1:500, Synaptic systems #197 004) to label axons from the dentate gyrus (mossy fibers). Sections were washed three times in PBS and incubated in combinations of the following secondary antibodies at 1:500 for 40 min at room temperature: streptavidin Alexa-488 (Invitrogen #S11226), goat anti-rabbit A633 (Invitrogen #A21071), goat anti-mouse A488 (Invitrogen # A11008), or goat anti-guinea pig A633 (Invitrogen #A21105). Sections were mounted with Vectashield antifade mounting medium with DAPI (Vector Laboratories #H-1500). Images were acquired on a Zeiss laser scanning confocal (LSM510 NLO) or a Zeiss light microscope using controlled camera settings. Images from the different wavelengths were pseudo-colored for figure clarity.

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Fluorescent Staining of Brain Cells

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Fluorescent staining of target proteins was performed as previously described (Peleg et al., 2010 (link)). In brief, mice were transcardially perfused with 4% PFA, brains isolated and post-fixed for another 16 h in 4% PFA. Free-floating cryosections (30 μm) were incubated with 5% goat serum for blocking and followed by incubation with target-specific primary antibodies (anti-NeuN [A60, MAB377, Merck Millipore, 1:1000, anti-GFAP [G5601, Promega, 1:1000], anti-IBA1 [019-19741, WAKO, 1:1000]). Corresponding secondary antibodies were from life Technologies (anti-mouse Alexa-488 labeled, A11029; anti-rabbit Alexa-633 labeled, A21071). Images were taken on a Leica SP2 confocal microscope. Stereological analysis of the number of cells was performed on 4 serial 40 μm free-floating coronal sections per animal which were analyzed by confocal microscopy to count cells expressing the indicated marker. Cell number was assessed as areal density across the CA1 region. The data was normalized to the 3 month groups.

Stilling R.M., Benito E., Gertig M., Barth J., Capece V., Burkhardt S., Bonn S, & Fischer A. (2014). De-regulation of gene expression and alternative splicing affects distinct cellular pathways in the aging hippocampus. Frontiers in Cellular Neuroscience, 8, 373.

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Immunocytochemical Visualization of Cell Adhesions

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Immunocytochemistry experiments were performed as described earlier (Faust et al., 2011 (link)). Primary antibodies used were anti-vinculin from mouse (V9131; Sigma), anti-vinculin from rabbit (700062; Invitrogen, Carlsbad, CA), anti-paxillin from mouse (AH00492; CellSignaling, Cambridge, UK), and anti-E-cadherin from mouse (610182; BD Bioscience, San José, CA). Alexa Fluor 488 anti-rabbit from chicken (A21441; Life Technologies, Carlsbad, CA), Alexa Fluor 488 anti-mouse from chicken (A21200; Life Technologies, Carlsbad, CA), Alexa Fluor 633 anti-rabbit from goat (A21071; Life Technologies), and Alexa Fluor 633 anti-mouse from goat (A21126; Life Technologies) were used as secondary antibodies. Actin was stained with Alexa Fluor 546 phalloidin (A22283; Life Technologies) and Alexa Fluor 488-i phalloidin (U0281; Abnova, Taipei City, Taiwan).

Noethel B., Ramms L., Dreissen G., Hoffmann M., Springer R., Rübsam M., Ziegler W.H., Niessen C.M., Merkel R, & Hoffmann B. (2018). Transition of responsive mechanosensitive elements from focal adhesions to adherens junctions on epithelial differentiation. Molecular Biology of the Cell, 29(19), 2317-2325.

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Optical Clearing and Immunolabelling of Brain Slices

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Optical clearing and immunolabelling brain slices were performed using the ABSOC method, as described in Muza et al.,.⁶² (link) In separate experiments, calretinin (CR), parvalbumin (PV), and neuropeptide-Y positive cells were immunolabelled using rabbit anti-calretinin (Abcam – ab244299, 1:5000), rabbit anti-parvalbumin (Abcam – ab11427, 1:5000), and rabbit anti-neuropeptide-Y (Abcam – ab10980, 1:5000) antibodies, respectively. All primary antibodies were probed with goat anti-rabbit Alexa Fluor 633 (ThermoFisher – A21071, 1:1000) secondary antibodies.

Muza P.M., Bush D., Pérez-González M., Zouhair I., Cleverley K., Sopena M.L., Aoidi R., West S.J., Good M., Tybulewicz V.L., Walker M.C., Fisher E.M, & Chang P. (2023). Cognitive impairments in a Down syndrome model with abnormal hippocampal and prefrontal dynamics and cytoarchitecture. iScience, 26(2), 106073.

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