Alexa fluor 633 goat anti mouse

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 633 goat anti-mouse is a fluorescently labeled secondary antibody used for detection and quantification of mouse primary antibodies in various immunoassays and imaging applications. The Alexa Fluor 633 dye has an excitation maximum at 632 nm and an emission maximum at 647 nm, making it compatible with standard red fluorescent protein detection methods.

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33 protocols using alexa fluor 633 goat anti mouse

Immunofluorescence Staining of Cellular Markers

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Cells cultured on glass coverslip were fixed with 4% PFA, treated with 0.1 M glycine in PBS for 30 min, followed by permeabilization with 0.2% saponin in PBS for 30 min and blocked with 5% goat or donkey serum and 0.05% BSA in 0.04% saponin containing PBS [64]. Coverslips were incubated overnight with primary antibodies at 4ºC and then with secondary antibodies in the blocking solution for 2 h at room temperature. Nuclei were stained with DAPI.
Primary antibodies include: SQSTM1 (1:200; Progen, GP62-C), TUBB3/Tuj1 (1:500; Biolegend, 801202), CTSL (1:250, R&D Systems, AF-1515), LAMP1 (1:200; Abcam, 24170). Secondary antibodies: Alexa Fluor 488 goat anti-rabbit (A11034), Alexa Fluor 546 anti-rabbit (A11035), Alexa Fluor 546 goat anti-mouse (A11030), Alexa Fluor 568 goat anti-guinea pig (A11075), Alexa Fluor 633 goat anti-mouse (A21052) and Alexa Fluor 546 donkey anti-goat (A11056) were from Invitrogen.

Sarkar C., Jones J.W., Hegdekar N., Thayer J.A., Kumar A., Faden A.I., Kane M.A, & Lipinski M.M. (2019). PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma. Autophagy, 16(3), 466-485.

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Drosophila Neuromuscular Junction Analysis

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All stocks were cultured on standard molasses/maize meal and agar medium in plastic vials or bottles at 25°C. Larvae were reared on apple juice plates supplemented with molasses/maize meal and yeast as previously described [79 (link)]. Larvae were selected for NMJ analysis at 5 days post egg laying. For analysis of bouton number was performed on the NMJ innervating muscles 6 and 7 from hemisegment A2 (1). Over 15 larvae were analysed for each genotype. For immunohistochemistry larvae were fixed for 20mins in 4% paraformaldehyde, or Bouin’s fixative for 30 minutes (GluRIIA). Primary antibodies used were anti-discs large (DLG, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa, USA),anti-Fas2 (DSHB), anti-GluRIIA (DSHB) and anti-BRP (DSHB), all used at 1/100. Secondary antibodies used were AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse (Invitrogen) at 1/1000, and anti-HRP-TRITC (The Jackson Laboratory, Bar Harbor, Maine, USA). Z-stacks were taken using a laser-scanning confocal microscope (Leica TCS SP5 II confocal microscope) and analysis performed using ImageJ and Adobe Photoshop. For statistical analysis of the genetic interactions, ANOVA was performed between the control, the two single heterozygous mutations and the transheterozygotes.

Grice S.J., Liu J.L, & Webber C. (2015). Synergistic Interactions between Drosophila Orthologues of Genes Spanned by De Novo Human CNVs Support Multiple-Hit Models of Autism. PLoS Genetics, 11(3), e1004998.

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Larval NMJ Developmental Analysis

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Larvae were reared, dissected and processed as previously described with analysis of hemisegment A2 (Schuster et al., 1996 (link); Grice et al., 2015a (link),b (link)), and at least 23 NMJs per genotype per stage were scored. Ectopic branch contact analysis was performed using L3 larval hemisegments A1 to A3, and 14 flies per genotype were scored. For axonal bruchpilot (Brp) localization studies, the transverse nerve (TN) section bypassing muscle 6 and 7 in hemisegments A2 and A3 was used. For immunohistochemistry, anti-discs large (DLG, DSHB), anti-HRP (Jackson), anti-Bruchpilot (DSHB) and anti-HA (Santa Cruz) were all used at 1/100 in combination with secondary antibodies, AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse (Invitrogen) at 1/1000. Z-stacks were taken using a Leica SP5 laser confocal microscope and analysis performed using ImageJ and Photoshop (Adobe).

Grice S.J., Sleigh J.N, & Zameel Cader M. (2018). Plexin-Semaphorin Signaling Modifies Neuromuscular Defects in a Drosophila Model of Peripheral Neuropathy. Frontiers in Molecular Neuroscience, 11, 55.

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Immunofluorescent Analysis of Ovarian Proteins

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Dissected ovaries and reproductive tracts of sacrificed adult female mice were fixed in 4% paraformaldehyde in PBS for 20 min and cryoprotected in 20% sucrose in PBS. Tissue sections with a thickness of 10 μm were cut from frozen specimens embedded in tissue freezing medium Jung (Leica Microsystems). Cryosections were mounted on Superfrost plus slides (Thermo Scientific), dried overnight, permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked for 60 min at room temperature with 1% BSA and 5% goat serum in PBS. Proteins were revealed by incubation with rabbit polyclonal anti-RIC8 (1:70, Proteintech Group, Inc) and anti-beta tubulin antibody (1:100, E7, Developmental Studies Hybridoma Bank) overnight at 4°C followed by Alexa Fluor 594 goat anti-rabbit (1:600, Invitrogen) or Alexa Fluor 633 goat anti-mouse (1:600, Invitrogen) secondary antibody for 60 min at room temperature. Cell nuclei were counterstained with DAPI and specimens mounted in Fluoromount G (Electron Microscopy Science). For negative controls, primary antibodies were omitted and no staining was observed.

Saare M., Lulla S., Tõnissoo T., Meier R., Kask K., Ruisu K., Karis A., Salumets A, & Pooga M. (2015). Expression Pattern and Localization Dynamics of Guanine Nucleotide Exchange Factor RIC8 during Mouse Oogenesis. PLoS ONE, 10(6), e0129131.

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Immunofluorescence Staining of Oocytes

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Oocytes and one-cell embryos were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100 for 20 min and blocked with 1% BSA in PBS for 1 h. Oocytes were stained as described above incubating with rabbit polyclonal anti-RIC8 antibody for 4 h at room temperature and using secondary antibody at dilution 1:800. Microfilaments were revealed with Alexa Fluor 488 phalloidin (1:125, Invitrogen), applied along with secondary antibodies.
For co-localization experiments, after staining with goat polyclonal RIC8 antibody (1:30; secondary antibody Alexa Flour 488 donkey anti-goat 1:800) and washes in PBS, oocytes were again blocked in 1% BSA-supplemented PBS for 1 h at room temperature, stained with rabbit polyclonal anti-NuMA (1:100, Abcam), rabbit polyclonal anti-LGN (1:100, Abcam) or mouse monoclonal anti-β-tubulin antibody for 4 h at room temperature. After washes in PBS, secondary antibody Alexa Fluor 555 goat anti-rabbit (1:800, Invitrogen) or Alexa Fluor 633 goat anti-mouse (1:800, Invitrogen) was added. After washes in PBS, oocytes were stained with DAPI (Sigma Aldrich) and mounted with Floromount (EMS). For negative controls primary antibodies were omitted and no staining was observed.

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Immunostaining Protocol for Neuronal Markers

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Before immunolabeling, dewaxed and rehydrated sections were blocked for 1 h with 1% Bovine Serum Albumin (BSA, cat nr: 9048‐46‐8, Merck) and 0.1% Triton X‐100 (Tx, cat nr: 9002‐93‐1, Sigma‐Aldrich) in phosphate‐buffered saline (PBS) pH 7.4. Then, slices were incubated overnight at 4°C with primary antibodies in PBS, 0.1% BSA, 0.001% Tx: anti‐β‐Tubulin Isotype III antibody (1:500, cat nr: 302302, Synaptic Systems); anti‐β‐Tubulin Isotype III antibody (1:500, cat nr: T5076, Sigma‐Aldrich); anti‐GAD67 (Gad1; 1:200, cat nr: MA5‐31377, Invitrogen); anti‐GluR2 (Gria2; 1:200, cat nr: PA5‐19496, Invitrogen); and anti‐CaMKIV (Camk4; 1:200, cat nr: PA1‐542, Invitrogen), respectively. Subsequently, slices were incubated for 2.5 h at RT with secondary antibodies in PBS, 0.1% BSA (1:2000 AlexaFluor 633 goat anti‐mouse cat nr: A21046 and 1:2000 AlexaFluor 488 goat anti‐rabbit, cat nr: A11034, Invitrogen; 1:2000 AlexaFluor 633 goat anti‐rabbit, cat nr: A21071, Invitrogen and 1:2000 AlexaFluor 488 goat anti‐mouse, cat nr: A11001, Invitrogen), respectively. Finally, glass slides were mounted with Fluoroshield with DAPI (cat nr: F6057, Sigma‐Aldrich). Between each step of the immunostaining procedure, sections were washed with PBS (3 × 10 min, RT).

Drulis‐Fajdasz D., Krzystyniak A., Puścian A., Pytyś A., Gostomska‐Pampuch K., Pudełko‐Malik N., Wiśniewski J.Ł., Młynarz P., Miazek A., Wójtowicz T., Włodarczyk J., Duś‐Szachniewicz K., Gizak A., Wiśniewski J.R, & Rakus D. (2023). Glycogen phosphorylase inhibition improves cognitive function of aged mice. Aging Cell, 22(9), e13928.

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3D Cell and Neuron Staining Protocol

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All the incubation steps in this staining protocol are performed on a plate shaker (at 30 rpm) unless stated otherwise. Encapsulated cells in 3D gels are washed twice with PBS for 30 minutes and are fixed with 4% paraformaldehyde (PFA) in PBS for 1 h on a plate shaker. After fixing, the gels are washed twice with PBS for 1 h and treated with 0.1% TritonX-100 for 40 minutes, and further washed twice with PBS for 1 h. For L929 fibroblasts, the gels are treated with iFluor-phalloidin 488 or 594 (Abcam, Germany) (1:1000) in PBS in combination with DAPI (1:100) and incubated for 4 h. The gels are washed twice with PBS for 1 h each and stored until imaging. The encapsulated DRGs in the gel are blocked with 5% bovine serum albumin (BSA) (Sigma, Germany) in PBS for 4 h, followed by the addition of primary beta-tubulin antibody TUJ1 (BioLegend, USA) (1:250) in 5% BSA solution for 18 h. The gels are washed twice with PBS for 1 h before adding the secondary antibody Alexa Fluor 633 goat anti-mouse (Invitrogen, Germany) (1:1000) in PBS and DAPI (1:100). The secondary antibody solution is incubated for 4 h and washed twice for 1 h each and stored in PBS until imaging with confocal microscopy.

Vedaraman S., Bernhagen D., Haraszti T., Licht C., Castro Nava A., Omidinia Anarkoli A., Timmerman P, & De Laporte L. (2021). Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels. Biomaterials Science, 9(12), 4329-4342.

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Immunofluorescence Staining for DNA/RNA Damage

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The sections were boiled for 3 min in citrate buffer (10 mmol/L, pH 6.0) at 95 °C to unmask epitopes, followed by blocking with 1% (w/v) bovine serum albumin (in PBS with 0.01% (v/v) Tween 20) for 30 min. Primary and secondary antibodies were diluted in PBS containing 0.02% (v/v) Triton X-100 and 0.02% (v/v) Tween 20. Between every protocol step, slides were washed in PBS 0.02% (v/v) and Tween 20 three times for 5 min. After application of the primary antibody DNA/RNA Damage Ab (15A3) (NB110-96878SS) (1:1000) overnight at 4 °C, sections were incubated for 1 h with the secondary antibody (1:500, AlexaFluor^®633 goat anti mouse, Invitrogen, Paisley, UK) at room temperature. Finally, the slides were mounted with fluorescent-free mounting medium (Prolong Gold antifade reagent, P36930, Life Technologies, UK). Maximal intensity projections were obtained with an inverted Zeiss LSM confocal microscope using Axiovision 4.8.2 SP 3 software with same settings for each slide.

Platt E., Robertson F., Al-Rashed A., Klootwijk R., Hall A., Quaglia A., Salama A., Heptinstall L, & Davidson B. (2024). NGAL in the Development of Acute Kidney Injury in a Murine Model of Remote Ischaemic Preconditioning and Liver Ischaemia Reperfusion. International Journal of Molecular Sciences, 25(10), 5061.

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Fluorescent Protein Staining Protocol

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The primary antibody solution contained chicken anti-GFP (1:1000, Abcam) and mouse anti-Bruchpilot (1:30, Developmental Studies Hybridoma Bank, nc82). The secondary antibody solution contained Alexa Fluor 488 goat anti-chicken (1:250, Invitrogen) and Alexa Fluor 633 goat anti-mouse (1:250, Invitrogen).

Joubès J., Phan T.H., Just D., Rothan C., Bergounioux C., Raymond P, & Chevalier C. (1999). Molecular and Biochemical Characterization of the Involvement of Cyclin-Dependent Kinase A during the Early Development of Tomato Fruit. Plant Physiology, 121(3), 857-869.

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Drosophila Neuromuscular Junction Imaging and Analysis

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Larvae were reared and dissected, as previously described (54 ), and at least 20 NMJs (muscles 6–7, hemisegment A2) per genotype per stage were scored (55 (link)). For immunohistochemistry, anti-discs large (DLG, DSHB, 4F3), anti-HRP (Jackson Immuno, 323-005-021), anti-bruchpilot (DSHB, nc82) and anti-HA (Santa Cruz, sc-805) were all used at 1/100, with AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse (Invitrogen) secondary antibodies at 1/1000. Z-stacks were taken using a Leica SP5 laser confocal microscope and analyses performed using ImageJ and Photoshop (Adobe). For BRP analysis in the axon, optical sections of 0.2 µm were taken using a laser-scanning confocal microscope (Leica TCS SP5 II confocal microscope). Bouton size analysis was performed by averaging two perpendicular diameter measurements of individual Ib boutons. All observable Ib boutons, which were identified by intense DLG staining, were measured per NMJ. All digital analyses and fluorescence measurements were performed using ImageJ. For BRP and HA staining at the synapse, average fluorescence intensity was analysed over the whole synapse (marked by HRP staining), again using optical sections of 0.2 µm. For HA staining, this was normalized to the background fluorescence intensity in the muscle.

Grice S.J., Sleigh J.N., Motley W.W., Liu J.L., Burgess R.W., Talbot K, & Cader M.Z. (2015). Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology. Human Molecular Genetics, 24(15), 4397-4406.

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