A32790

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A32790 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the A32790 is to perform various tasks and procedures in a laboratory setting. Further details about the specific capabilities and intended use of this product are not available.

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Lab products found in correlation

20 protocols using a32790

Longitudinal Tibia Analysis of Bone Development

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Mice were sacrificed on PSD 3, 7, and 10. Thick (90 μm) longitudinal tibia frozen sections were prepared as described previously (Kusumbe et al., 2015 (link)). In brief, each tibia was stripped of soft tissues and fixed in 4% paraformaldehyde (PFA) at 4°C for 4 h, decalcified in 0.5 M EDTA at 4°C for 48 h, cryoprotected at 4°C for 48 h, and cryoembedded. Cryosections were prepared using a cryostat (Leica 3050S) with a thickness of 80 μm and stored at −80°C.
For immunostaining, the tibia sections were hydrated, permeabilized, blocked, and incubated with primary antibodies against endomucin (sc-65495, 1:100, Santa Cruz), osterix (ab22552, 1:200, Abcam), paired related homeobox 1 (PRRX1) (ab211292, 1:200, Abcam), KI67 (ab15580, 1:200, Abcam), and F4/80 (ab6640, 1:200, Abcam) overnight. Next day, the sections were incubated with anti-rabbit Alexa Fluor 488 (A32790, 1:300, Invitrogen) secondary antibody for 1 h at room temperature. Sections were washed with PBS and mounted with DAPI FluoroMount-G (0100-20, SouthernBiotech) and then sealed with coverslips.

Yang C., Li Z., Liu Y., Hou R., Lin M., Fu L., Wu D., Liu Q., Li K, & Liu C. (2022). Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair. Stem Cell Reports, 17(10), 2318-2333.

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Immunofluorescence Staining of Cathepsin and Iron Markers

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The injured CAs underwent a series of processes, including excision, fixation, and embedding in paraffin. Then, the CAs were cut into 4 μm thick sections and analyzed through IF staining. Homoplastically, the treated HUVECs were fixed in 4% paraformaldehyde. Next, as previously described [20 (link)], the sections and HUVECs were incubated with primary antibodies against cathepsin B (1 : 100, ab214428, Abcam), cathepsin D (1 : 200, #2284 s, Cell Signaling Technology), ferritin (1 : 50, ab75973, Abcam), and TfR (1 : 50, ab269513, Abcam) at 4°C overnight. After washing three times on the following day, the samples were then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32790, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32794, Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (1 : 300, A-11001, Invitrogen), and Alexa Fluor 555-conjugated goat anti-mouse (1 : 300, A-21424, Invitrogen) secondary antibodies at 37°C for 1 h. Next, 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA) was added to each section for coverslipping. Finally, the sections were observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan), and ImageJ software was used to quantify the fluorescence intensity.

Wang Y., Bao D., Dong Y., Wei X., Yu J, & Niu C. (2022). α-Lipoic Acid-Plus Ameliorates Endothelial Injury by Inhibiting the Apoptosis Pathway Mediated by Intralysosomal Cathepsins in an In Vivo and In Vitro Endothelial Injury Model. Oxidative Medicine and Cellular Longevity, 2022, 8979904.

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Immunofluorescence Analysis of Popliteal Arteries

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Popliteal arteries were fixed with 4% paraformaldehyde for 24 h at room temperature, followed by procession by a serial alcohol gradient and embedment in paraffin wax blocks. The slides were incubated with primary antibodies that include GAB1 Rabbit mAb (#3232; Cell Signaling), LC3B (E5Q2K) Mouse mAb (#83506; Cell Signaling), and Human CD31/PECAM-1 Antibody Sheep mAb (#AF806-SP; R&D) overnight at 4°C, followed by the incubation of secondary antibodies that include donkey anti-rabbit IgG Alexa 488 (#A32790, Invitrogen, green), donkey anti-mouse IgG Alexa plus 647 (#A32787, Invitrogen, pink), and donkey anti-Sheep IgG Alexa 555 (#A-21436, Invitrogen, red). Nuclei were stained with DAPI (#D1306; Invitrogen, Paisley, United Kingdom). An epifluorescence microscope (#DM4000 M; Leica, Wetzlar, Germany) was used for image capture and analysis.

Qian X., Wang H., Wang Y., Chen J., Guo X, & Deng H. (2021). Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression. Frontiers in Physiology, 11, 559396.

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Dual-Labeling Immunofluorescence of DRG Neurons

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Double-label immunofluorescence immunohistochemistry was carried out as described in our previous reports (Jahan et al., 2015 (link); Islam et al., 2017 (link)). In brief, free floating sections of DRG (cervical to sacral) were blocked with 10 % NDS containing 0.3 % Triton X-100 at 4 °C for 2−3 h and incubated with goat anti-HAP1 (1:10,000) antibody in combination with a rabbit anti-SP (1:1,000), rabbit anti-CGRP (1:1,000), rabbit anti-CB (1:20,000), rabbit anti-NOS (1:1000), rabbit anti-TRPV1 (1:1000), rabbit anti-CR (1:1,000), rabbit anti-PV (1:1,000), rabbit anti-TH (1: 1,000), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1; 1: 1,000) or rabbit anti-neuronal nuclei (NeuN; 1: 1,000) antibody at 20 °C for 5 d (Table 1 for detail about primary antibodies). After washing three times the sections were incubated with a mixture of Alexa Fluor 594-conjugated donkey anti-goat IgG (A11058, AB_2534105, Invitrogen, Eugene, OR, USA; 1:1,000) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A32790, AB_2762833, Invitrogen, Rockford, IL, USA; 1:1,000) secondary antibodies at 20 °C for 2−3 h. After washing three times with PBS, the sections were then mounted on glass slides, air-dried for 30 min and finally embedded with Fluoromount/Plus (K048, Diagonostic Biosystems, Pleasanton, CA, USA).

Islam M.N., Maeda N., Miyasato E., Jahan M.R., Tarif A.M., Ishino T., Nozaki K., Masumoto K.H., Yanai A, & Shinoda K. (2020). Expression of huntingtin-associated protein 1 in adult mouse dorsal root ganglia and its neurochemical characterization in reference to sensory neuron subpopulations. IBRO Reports, 9, 258-269.

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Immunofluorescence Staining Protocol

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Cells were seeded in chamber well slides (Millipore Millicell EZ Slides) and cultured overnight. The following day, cells were treated with compounds for various durations. Cells were then fixed and stained as described previously (41 (link)). Briefly, cells were fixed with 1% paraformaldehyde/2% sucrose for 15 minutes at room temperature, 100% methanol for 30 minutes at −20°C, and then 50% methanol/50% acetone for 20 minutes at −20°C. Cells were then incubated in permeabilization/blocking solution (Cell Signaling Technology #12411) at room temperature for 1 hour. Primary antibodies (Cell Signaling Technology #9718 or #80312 for γH2Ax, #2675 for phospho-53BP1, and #86298 for beta-tubulin) were diluted 1:250 in permeabilization/blocking solution and incubated with the cells at 4°C overnight. The secondary antibodies used were Alexa Fluor 488 (Invitrogen #A32766 donkey anti-mouse IgG or #A32790 donkey anti-rabbit IgG) and Alexa Fluor 555 (Invitrogen #A32773 donkey anti-Mouse IgG or #A32794 donkey anti-rabbit IgG). Three washes with PBS with Triton X-100 and four washes with PBS were performed after primary and secondary antibody. Cells were costained with DAPI (Invitrogen #D1306) to visualize the nuclei and micronuclei. Slides were imaged on a Zeiss MIC-074 microscope (Carl Zeiss Microscopy, LLC). Images were processed and analyzed using ImageJ software.

Turchick A., Zimmermann A., Chiu L.Y., Dahmen H., Elenbaas B., Zenke F.T., Blaukat A, & Vassilev L.T. (2023). Selective Inhibition of ATM-dependent Double-strand Break Repair and Checkpoint Control Synergistically Enhances the Efficacy of ATR Inhibitors. Molecular Cancer Therapeutics, 22(7), 859-872.

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Immunofluorescence Staining of Murine Kidneys

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For immunofluorescence staining of kidneys from mice, fresh cut 4 μm-thick tissue in OCT was used. No fixation agent was applied. Permeabilization was performed using 0.3% Triton X-100 in 1 × PBS for 15 min at room temperature. A blocking step was performed using Power Block Universal Blocking Reagent (BioGenex, Fremont CA, USA) for 10 min at room temperature. Primary antibodies (for APE1 (NB100-101, rabbit, 1:500, NOVUS biologicals) and synaptopodin (sc-21537, goat, 1:500, Santa Cruz Biotechnolgy Inc.) were used for 24 h at 4 °C. Secondary antibodies (anti-rabbit Alexa Flour 488 (A32790, donkey, Invitrogen) and anti-goat Alexa Flour 568 (A11057, donkey, Invitrogen)) were used for 1 h at room temperature. The anti-caspase-11 antibody was conjugated with Alexa Flour 594 and used without secondary antibodies. To detect nucleus, ProLong Gold antifade reagent with DAPI (Invitrogen, St. Louis MO, USA) was applied.
Images were acquired by laser scanning confocal microscopy using a Leica SP5 Inverted microscope, × 20 objective (Leica Microsystems CMS GmbH, Germany). Measuring of cell fluorescence was performed using Image J software.

Ito M., Ducasa G.M., Molina J.D., Santos J.V., Mallela S.K., Kim J.J., Ge M., Mitrofanova A., Sloan A., Merscher S., Mimura I, & Fornoni A. (2023). ABCA1 deficiency contributes to podocyte pyroptosis priming via the APE1/IRF1 axis in diabetic kidney disease. Scientific Reports, 13, 9616.

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Immunohistochemical Analysis of Neuronal Markers

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Mice were anesthetized with sevoflurane, and brains were cut on a sliding freezing stage microtome in 30 μm thick sections. Hippocampal sections were washed 3 times in PBS. Following 15 min permeabilization with 0.3% Triton X-100 in 0.01 M PBS, sections were blocked with 10% donkey serum for 2 h. Next, sections were incubated overnight at 4°C with the following primary antibodies: DHX9 (1:300, 17721-1-AP, Proteintech), NeuN (1:200, ab104224, Abcam), Iba1 (1:200, ab5076, Abcam), GFAP (1:200, ab3554, Abcam), MAP2 (1:200, ab11267, Abcam), FMRP (1:100, sc-101048, Santa Cruz), STAU1 (1:100, sc-390820, Santa Cruz), or c-Fos (1:200, ab208942, Abcam). The next day, slides were washed 3 times for 7 min in PBS. Then, sections were incubated with the corresponding fluorescent-conjugated secondary antibody (A32766, A32744, A32790, A32754, Invitrogen) for 2 h at room temperature in the dark. DAPI Fluoromount-G aqueous mounting medium with anti-fade properties (SouthernBiotech) was used to identify cell nuclei. Slides were stored in the dark at 4°C until needed for analysis. Fluorescence images were captured with an Olympus FV1000 laser confocal microscope. For c-Fos positive neuron analysis, ImageJ was used to analysis the signals of hippocampal neurons from 3 animals per condition.

Yang L., Liu Q., Zhao Y., Lin N., Huang Y., Wang Q., Yang K., Wei R., Li X., Zhang M., Hao L., Wang H, & Pan Z. (2024). DExH-box helicase 9 modulates hippocampal synapses and regulates neuropathic pain. iScience, 27(2), 109016.

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Immunohistological Characterization of Human Dental Pulp

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Cryosections of human dental pulps were washed with PBS, incubated with blocking solution (1%BSA, 10% donkey serum, 0.5% Triton in PBS) for 45 min at room temperature, followed by incubation with the primary antibody at +4 °C, overnight. Primary antibodies used were as follows: ACTA2 (αSMA, ab5694, 1:200), COL1A1 (ab34710, 1:100), COL3A1 (ab6310, 1:200), CD31 (PECAM, ab9498, 1:100), LAMININ (ab11575, 1:100), DECORIN (ab175404, 1:100), TENASCIN C (LS-B8789/74286, 1:50). After incubation with the primary antibody, sections were extensively washed and incubated with appropriate secondary antibody (1:500) for 45 min at room temperature. The secondary antibodies used include donkey anti-rabbit (AF488, A32790, Invitrogen) and donkey anti-mouse (AF647, A31571, Invitrogen). After incubation, slides were washed with PBS, mounted using Vectashield with DAPI, and imaged with Leica STELLARIS 5 automated upright confocal laser scanning microscope equipped with HC PL APO CS2 20x/0.75 NA Air and HC PL APO CS2 63x/1.3NA Glycerol objectives.

Balic A., Perver D., Pagella P., Rehrauer H., Stadlinger B., Moor A.E., Vogel V, & Mitsiadis T.A. (2023). Extracellular matrix remodelling in dental pulp tissue of carious human teeth through the prism of single-cell RNA sequencing. International Journal of Oral Science, 15, 30.

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Immunofluorescence Analysis of Cathepsin, Ferritin and TfR

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The injured CA was excised, xed, embedded in para n and cut into 4-µm sections and examined by IF staining. Homoplastically, the disposed HUVECs were xed by 4% paraformaldehyde. As previously described [20] , The sections and HUVECs were incubated with primary antibodies to Cathepsin B (1:100, ab214428, abcam), Cathepsin D (1:200, #2284s, Cell Signaling Technology), Ferritin (1:50, ab75973, abcam) and TfR (1:50, ab269513, abcam) at 4°C overnight. Then, it was incubated with the Donkey anti-
Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:300, A32790, Invitrogen) and Alexa Fluor 555 (1:300, A32794, Invitrogen) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:300, A-11001, Invitrogen) and Alexa Fluor 555 (1:300, A-21424, Invitrogen) at 37°C for 1 hour after washed 3 times on the following day. Next, the sections were added into 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA) for coverslipping. In the end, the sections were observed under a uorescence microscopeOLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan, and ImageJ software was used for quantizing the uorescence intensity.

Wang Y., Kong X., Bao D., Xu B., Dong Y., Wei X., & Niu C. (2021). α -Lipoic Acid-Plus Ameliorates Endothelial Injury via Inhibiting the Apoptosis Pathway Mediated by Intralysosomal Cathepsins in Vivo and Vitro Endothelial Injury Model.

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Quantifying Focal Adhesion Formation

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To quantify the formation of focal adhesions, the cells were preincubated in culture medium containing 10 mM GlcNAc and HAse-treated control cells were created as described above. The cells were seeded on 8-well ibidiTreat polystyrene chamber slides in densities of 5000 and 10,000 cells/cm². The slides were used uncoated as well as coated with 5 µg/cm² Col-I (Merck, Darmstadt, Germany) overnight at 4 °C. 20, 40 and 90 min after seeding, the cells were washed two times with DPBS and fixed with 4% paraformaldehyde in DPBS. Cells seeded to the 8-well slides were stained like for confocal microscopy as mentioned above, only including an additional permeabilization step after fixation with 300 µL 0.1% Triton X100 (Sigma-Aldrich) in DPBS per well. As primary antibody, a rabbit anti-Vinculin (1:250, ab129002, Abcam) and as secondary antibody a donkey anti-rabbit Alexa Fluor 400 conjugate (4 µg/mL, A32790, Thermo Fisher Scientific) were used. The nuclei were counterstained with DAPI and the slides were covered with mounting medium (Ibidi). The experiment was performed twice. Per cell line, treatment and time point between 28 and 39 cells were imaged with an AxioObserver Z1 (Carl Zeiss) and the number of focal adhesions was counted using the FIJI software.

Reiprich S., Hofbauer E., Kiderlen S., Clausen-Schaumann H., Böcker W., Aszódi A, & Schönitzer V. (2020). Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells. International Journal of Molecular Sciences, 21(11), 3827.

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