The 293T cells were cultured at coverslips and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT, and fixed by 4% PFA for antibody immunofluorescence staining after 24 h of transfection. The samples were incubated in primary antibodies (1:500) for 3 h and secondary antibodies conjugated with Alexa Fluor 647 (1:500) for 2 h, with 15 min DAPI staining before being mounted in Prolong gold antifade reagent (ThermoFisher Scientific, P36934). For SiRNeoblasts, 20,000 cells were sorted out for each group and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT. At 24 hpt, following fixed by 4% FA in 0.4X PBS twice for 10 min, incubated in Hybe at 56°C for 2 h and blocked by 10% Horse serum in PBSTx0.1% at room temperature for 30 min, the cells were stained with primary antibodies (1:500) and secondary antibodies conjugated with Alexa Fluor 555 (1:500) for 2 h, respectively. The antibodies included rabbit polyclonal mCherry antibody (MBL, PM005), mouse monoclonal Flag antibody clone M2 (Sigma, F1804), goat anti-mouse IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150119), goat anti-rabbit IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150083), goat anti-mouse IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150118) and goat anti-rabbit IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150086).
Ab150118
Manufactured by Abcam
Sourced in United Kingdom
Ab150118 is a rabbit monoclonal antibody that targets the protein Vimentin. It is suitable for use in applications such as Immunohistochemistry, Western Blot, and Immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab150077, by Abcam (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Ab150119, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Ab150086, by Abcam (1 mentions)
Ab150083, by Abcam (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Ab150075, by Abcam (1 mentions)
Sp8 confocal microscopy, by Leica (1 mentions)
B2064, by Merck Group (1 mentions)
Ab9465, by Abcam (1 mentions)
Ab33589, by Abcam (1 mentions)
Ab228551, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab76055, by Abcam (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Ab150129, by Abcam (1 mentions)
Ab97077, by Abcam (1 mentions)
Ab25630, by Abcam (1 mentions)
13 protocols using ab150118
1
Protein Localization in Transfected Cells
2
Cardiomyocyte and Heart Tissue Immunostaining
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Heart section (5 µm) were subject to deparaffinization and rehydration followed by antigen retrieval by heating the slides in 10 mM Citrate buffer (pH 6.0) at 95 °C for 10 min. For cardiomyocytes, NRCMs cultured on glass coverslips were fixed in paraformaldehyde for 15 min at room temperature. After washing for three times with PBS each for 5 min, cardiomyocytes and heart sections were permeabilized with 0.5% (v/v) Triton X-100 for 20 min and then blocked with 5% (v/v) bovine serum albumin (BSA, Sigma-Aldrich, B2064) for 1 h at room temperature. Next, the samples were incubated primary antibodies of cTnT (Abcam, ab8295) or FGF20 (Santa Cruz sc-373927) at 4 °C overnight. After washing, samples for cTnT were incubated with Alexa fluor 647-conjugated anti-rabbit IgG secondary antibody (Abcam, ab150075), and samples for FGF20 were incubated with Alexa fluor 555-conjugated anti-mouse IgG secondary antibody (Abcam, ab150118). Finally, DAPI was used to label the cell nuclei. Images were acquired with a Leica SP8 confocal microscopy.
3
Cardiac Muscle Cell Immunostaining
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At
14 days of culture, samples were rinsed with PBS, and cells were fixed
with 4 wt % paraformaldehyde (PFA) at room temperature for 30 min.
After fixation, cells were permeabilized with 0.1% Triton X-100 for
5 min and blocked with 1 wt % bovine serum albumin containing 22.52
mg/mL glycine prepared in PBS for 30 min. Then, samples were incubated
in anti-sarcomeric α-actinin (1:50 dilution, ab 9465, Abcam)
and monoclonal anti-cardiac troponin T (cTnT) (1:25 dilution, ab33589,
Abcam) primary antibodies for 4 h at room temperature.41 (link) Afterward, samples were rinsed with PBS and
incubated in Alexa Fluor 555 (1:1000, ab150118, Abcam) and Alexa Fluor
488 (1:1000, ab150113, Abcam) secondary antibodies for 1 h to stain
α-actinin and cTnT, respectively. The actin cytoskeletons of
the cells were stained with Alexa Fluor 488 Phalloidin (Thermo Fisher).
The nuclei of cells were counterstained with 25 mM Hoechst (Abcam,
ab228551). Images were acquired using a confocal microscope (DMI 8,
Leica), and processed using Leica LASX software.
14 days of culture, samples were rinsed with PBS, and cells were fixed
with 4 wt % paraformaldehyde (PFA) at room temperature for 30 min.
After fixation, cells were permeabilized with 0.1% Triton X-100 for
5 min and blocked with 1 wt % bovine serum albumin containing 22.52
mg/mL glycine prepared in PBS for 30 min. Then, samples were incubated
in anti-sarcomeric α-actinin (1:50 dilution, ab 9465, Abcam)
and monoclonal anti-cardiac troponin T (cTnT) (1:25 dilution, ab33589,
Abcam) primary antibodies for 4 h at room temperature.41 (link) Afterward, samples were rinsed with PBS and
incubated in Alexa Fluor 555 (1:1000, ab150118, Abcam) and Alexa Fluor
488 (1:1000, ab150113, Abcam) secondary antibodies for 1 h to stain
α-actinin and cTnT, respectively. The actin cytoskeletons of
the cells were stained with Alexa Fluor 488 Phalloidin (Thermo Fisher).
The nuclei of cells were counterstained with 25 mM Hoechst (Abcam,
ab228551). Images were acquired using a confocal microscope (DMI 8,
Leica), and processed using Leica LASX software.
4
Immunofluorescence Staining of E-cadherin
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The sections of avian IO were washed with Tris-buffered saline (TBS ) containing 0.1% Tween 20 (TBST ) and blocked using 10% normal goat serum (Vector Laboratories) in TBST for one h at room temperature, incubated with mouse anti E-cadherin (1:250, ab76055, Abcam, Waltham, MA) overnight at 4°C. After 3 washes with TBST, the secondary antibody Alexa Fluor 555 goat antimouse IgG (1:1000, ab150118, Abcam) was added and incubated at room temperature for one h followed by 3 times washed with TBST. The stained slides were mounted with one drop of Prolong Diamond Antifade DAPI (Thermo Fisher Scientific,Waltham, MA; P36966) and overlaid with a cover glass. Images were captured by all-in-one BZ X800 Keyence Fluorescence Microscope.
5
Immunocytochemistry of iNSC and 293T Cells
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iNSC-Spon1 and 293T-APP-SPON1 cells were fixed by 4% paraformaldehyde for 15 min, and then 1% Triton-X was treated for permeabilization. Next, cells were incubated with 4% FBS for 30 min. Primary antibodies that were diluted by 4% FBS were added for 1 h at room temperature (RT). After washing, secondary antibodies (1:1000) diluted in PBS were added for 1 h at RT. Secondary antibodies included Alexa488 anti-goat antibody (ab150129, abcam, Cambridge, UK), which was used for V5 and Sox2 staining, and Alexa555 anti-mouse antibody (ab150118, abcam, Cambridge, UK), which was used for Nestin staining. Cells were counterstained by Hoechst 33,342 (Invitrogen, Carlsbad, CA, USA). The cells were imaged using a Leica fluorescence microscopy (DMI 3000 B, Leica, Wetzlar, Hesse, Germany). The primary antibodies are listed in Supplementary Table S2 .
6
Visualizing Mitophagy via Confocal Microscopy
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To visually assess mitophagy, cells seeded on coverslips were fixed with 4% paraformaldehyde in PBS, and immunostained with anti-LAMP1 mouse monoclonal antibody (ab25630, Abcam) and anti-Tom20 rabbit polyclonal antibody (sc-11415, Santa Cruz Biotech). Goat anti mouse IgG AlexaFlour555 (ab150118, Abcam) and goat anti-rabbit IgG Cy5 (ab97077, Abcam) were used as secondary antibodies. Morphology and colocalization of mitochondria with lysosomes were assessed using a CLSM Leica SP5 Confocal Laser Scanning Inverted Microscope (Leica Microsystems) equipped with a helium neon laser. The objective used was oil immersion ×63/1.4. Plan Apo.
Colocalization of LAMP1 and TOM20 was quantified using Mander’s colocalization coefficient on ImageJ (COLOC2 plugin).
Colocalization of LAMP1 and TOM20 was quantified using Mander’s colocalization coefficient on ImageJ (COLOC2 plugin).
7
Immunolabeling of Synaptonemal Complexes and Chromatin Markers
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Slides were washed with phosphate-buffered saline (PBS) and were incubated with primary antibodies that had been diluted in the antibody dilution buffer (ADB: 3% bovine serum albumin (BSA), 0.05% Triton X-100 in PBS) at 4 °C overnight. SCs were detected using rabbit polyclonal antibodies against the SC axial element protein SYCP3 (ab15093, 1:250, Abcam, Cambridge, UK), centromeres were detected using human anti-centromere antibodies ACA (1:500; Antibodies Incorporated 15–234, Davis, CA, USA), and the inactivated chromatin of the sex bivalents was stained with the mouse monoclonal anti-γH2AX antibodies (ab26350, 1:800, Abcam, Cambridge, UK). After washing, the corresponding secondary antibodies that had been diluted in ADB were used: goat anti-rabbit Immunoglobulin (Ig) G, Alexa Fluor 555 (ab150078, 1:500, Abcam, Cambridge, UK), goat anti-rabbit Immunoglobulin (Ig) G, Alexa Fluor 488 (ab150077, 1:500, Abcam, Cambridge, UK), goat anti mouse Immunoglobulin (Ig) G, Alexa Fluor 555 (ab150118, 1:500, Abcam, Cambridge, UK), and goat anti-human Alexa Fluor 488 (A-11013, 1:500, ThermoFischer, Waltham, MA USA). Secondary antibody incubations were performed in a humid chamber at 37 °C for 2 h.
8
Immunohistochemical Analysis of Acox1 and α-SMA in Renal Tubular Epithelial Cells
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All the specimens were deparaffinized and rehydrated. Following antigen retrieval, the tissues were incubated with primary anti-Acox1 (1:100, 10957-1-AP; Proteintech group) and α-SMA (1:200, BM0002; Boster Biotechnology) antibodies at 4 °C overnight. At least ten fields were randomly selected to evaluate the percentage of Acox1-positive and α-SMA-positive RTECs. For immunofluorescence, all sections were incubated with anti-Acox1 antibody (1:100, 10957-1-AP; Proteintech group) followed by Alexa-488 conjugated goat anti-rabbit antibody (1:200, ab150077; Abcam), anti-α-SMA antibody (1:200, BM0002; Boster Biotechnology), and anti-collagen I (1:100, ab6308; Abcam) followed by Alexa-555 conjugated goat anti-mouse antibody (1:200, ab150118; Abcam) at 4 °C overnight. The cells were then co-stained with DAPI (1:500, C1006; Beyotime Biotechnology). All samples were analyzed using a fluorescence microscope (Leica, Germany). At least six fields were randomly selected to evaluate the percentage of positive staining with Image J software (version 1.37; National Institutes of Health, Bethesda, MD).
9
Immunofluorescence Staining of Cell Cultures
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The samples were washed twice with cold PBS after removing the culture medium. After that, the cells were fixed using 4% PFA in PBS for 20 min at room temperature and then washed three times with PBS. To permeabilize the samples, 0.1% Triton-X100 in PBS was added for 5 min, followed by three more washes with PBS. Next, the samples were treated with a blocking solution (2% BSA in PBS) for 1 h at room temperature. Primary antibodies were then diluted in a blocking solution according to the specifications shown in Table 1 , and then they were added to the samples for 1 h at room temperature or at 4 °C overnight. The following secondary antibodies were incubated for 1.5 h: goat anti-rabbit Alexa 488 (Abcam, ab150077, 1:250), goat anti-mouse Alexa 647 (Abcam, ab150119, 1:250), and goat anti-mouse Alexa 555 (Abcam, ab150118). Phalloidin staining was performed by adding phalloidin conjugated to iFluor 647 (Abcam, ab176759, 1:1000) or phalloidin conjugated to iFluor 555 (Abcam, ab176756, 1:1000). For nuclei detection, the samples were incubated for 5 min with Hoechst 33258 (1:20; Sigma). Images were taken using confocal microscopy (Nikon Eclipse Ni, Tokyo, Japan) or an inverted fluorescence microscope (Nikon Eclipse TI).
10
Visualizing 5-HT2A Receptor and Macrophages
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For the human tissues, 4-μm-thick frozen sections of intestinal specimens were fixed in cold acetone for 10 min at −20°C and blocked with 5% BSA for 1 h at room temperature, then incubated with a primary rabbit anti-human 5-HT2AR antibody (ab66049; rabbit polyclonal to 5-HT2AR; reacts to mouse, rat, human; Abcam, Cambridge, UK; 1:500) overnight at 4°C. Mouse anti-human CD68 antibody (MCA5709; mouse anti-human CD68, monoclonal antibody; AbD Serotec, Kidlington, UK; 1:500) (overnight at 4°C) was subsequently used to detect the macrophages. For the murine colon tissues, 5-HT2AR expression was detected by overnight incubation at 4°C with rabbit anti-mouse 5-HT2AR antibody (ab66049; rabbit polyclonal to 5-HT2AR; reacts to mouse, rat, human; Abcam; 1:300). Subsquently, rat anti-mouse CD68 antibody (MCA1957GA; rat anti-mouse CD68, monoclonal antibody; AbD Serotec; 1:500) was used overnight at 4°C. For both analyses, Alexa-Fluor 488- and Alexa-Fluor 555-conjugated antibodies [goat anti-rabbit IgG (ab150077, goat anti-rat IgG (ab150158) and goat anti-mouse IgG (ab150118); all from Abcam, Cambridge, UK; 1:1,000; 1 h at room temperature] were used as secondary antibodies, followed by incubation with 1 μg/ml DAPI (20 min, room temperature). The sections were finally visualized under a confocal microscope (Olympus, Tokyo, Japan). Images were captured using FluoView software.
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