Paraffin sectioned and cryosectioned tissue was subjected to haematoxylin and eosin staining or immunofluorescence. For immunofluorescence assays, tissues were fixed in 4% PFA, paraffin embedded and sectioned at 5 μm, or embedded in optimal cutting temperature embedding medium and cryosectioned at 10 μm. Paraffin sections were de-waxed through graded xylene solutions, microwaved in 10 mM sodium citrate and incubated with primary antibodies, followed by incubation with Alexa Fluor-labelled secondary antibodies (Thermo Fisher Scientific)16 (link). For BrdU labelling, mice were injected with 50 μg BrdU g−1 body weight and killed after 1–2 h. Skin samples were fixed in 4% PFA, embedded in paraffin, de-waxed and incubated with anti-BrdU antibody (Rockland Immunochemicals, Limerick, PA, 600-401-C29, 1:100)16 (link). For tail skin whole mounts, skin samples were incubated with Dispase II and separated epidermis was counterstained with Hoechst 33342 (Thermo Fisher Scientific) and imaged on a Leica TCS SP8 confocal microscope (Leica Microsystems, Buffalo Grove, IL). For analyses of proliferation in slides stained for Ki67 or BrdU, sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Sectioned tongue and footpad tissues were subjected to co-immunofluorescence for KRT14 to identify basal cells.
Alexa fluor labelled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-fluor-labelled secondary antibodies are fluorescent-conjugated antibodies used in immunoassays and immunohistochemistry to detect and visualize target proteins. They bind to primary antibodies and produce a fluorescent signal upon excitation, allowing for the identification and localization of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Aquamount, by Polysciences (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti mcherry, by Abcam (2 mentions)
A1 rs scanning confocal microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Anti brdu antibody, by Rockland Immunochemicals (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Snap25, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 633 phalloidin, by Thermo Fisher Scientific (1 mentions)
Tunel assay kit, by Roche (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Uv meta 510 confocal microscope, by Zeiss (1 mentions)
α sma, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lsm 980 confocal microscope, by Zeiss (1 mentions)
Aperio scanscope xt, by Leica (1 mentions)
Mounting media, by Agilent Technologies (1 mentions)
Triton x 100, by Merck Group (1 mentions)
25 protocols using alexa fluor labelled secondary antibody
1
Immunofluorescence and Proliferation Assays for Skin Tissues
2
Immunofluorescent Neurite Analysis
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Cells grown on coverslips were fixed with 4% PFA (Sigma-Aldrich, 16005) and blocked in PBS containing 10% FBS (ThermoFisher, 10270-106) and 0.3% Triton X-100. Primary antibodies diluted in blocking solution were applied at 4 °C overnight. Target proteins were detected with Alexa Fluor-labelled secondary antibodies (ThermoFisher) diluted in blocking solution. Cell nuclei were stained with DAPI and coverslips were mounted on glass slides using Mowiol®/DABCO. DAPI + and NeuN+ nuclei were counted manually using Fiji. The total length of the neurite network per image was quantified using the ridge detection plugin for Fiji. The total neurite length in µm was divided by the number of cell nuclei to calculate the average neurite length per cell.
3
Immunofluorescence Staining of HSV-1 Capsids
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Rabbit antibody against purified HSV-1 nuclear C capsids (PTNC) (1:5500 dilution) was kindy provided by Dr. Frazer Rixon, (MRC Virology Unit, Institute of Virology, UK) [19 (link)]. Mouse monoclonal anti-non-muscle myosin IIB (1:100 dilution, ab684) was obtained from Abcam, UK. Goat polyclonal anti-myosin V (1:200 dilution, LS-C139627) was obtained from Lifespan Bioscience, USA. Rabbit polyclonal anti-kinesin family member 3A (KIF3A, 1:50 dilution, SC-50457), rabbit polyclonal anti-Rab6 (1:50 dilution, SC-310) and mouse monoclonal anti-synaptosome associated protein 25 (SNAP25, 1:20 dilution, SC-20038) were obtained from Santa Cruz, USA. Alexa Fluor 633 Phalloidin and Alexa Fluor-labelled secondary antibodies were obtained from Thermo Fisher Scientific, USA.
4
Histomorphometric Analysis of Knee Cartilage
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As described for the histomorphometric analysis, three frontal knee sections (5 μm) at 30 μm intervals (Fig 1A ) were analysed by immunostaining with primary antibodies against collagen type I (COL1 A1/A2; Acris), type II (COL2 A2 [C-19]; Santa Cruz) and collagen cleavage products (Col2 3/4Cshort; IBEX Pharmaceuticals). The collagen type I staining was used for discrimination of articular cartilage and osteophytic cartilage structures (not shown). The collagen type II staining was used to discriminate the cartilages non-calcified from the calcified layer. Incubation with primary antibodies was followed by incubation with corresponding AlexaFluor-labelled secondary antibodies (Invitrogen) and DAPI staining of cell nuclei. Apoptosis in cartilage tissues was determined by TUNEL assay kit according to the manufacturer’s protocol (Roche Diagnostics).
5
Immunofluorescence Staining of Mitochondria and Lysosomes
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Cover slips were fixed in phosphate-buffered saline buffer containing 4% paraformaldehyde at 37°C for 15 min followed by quenching with ammonium chloride (50 μM). Fixed cover slips were stained with an αTOMM20 antibody (sc-11415; Santa Cruz) for detection of mitochondria or an αLAMP1 antibody (sc-18821; Santa Cruz) for detection of lysosomes. The samples were then incubated with appropriate Alexa Fluor–labelled secondary antibodies (Invitrogen). Finally, the cover slips were mounted for imaging with Dako fluorescent mounting medium containing DAPI (S3023; Agilent Technology).
6
AdipoRon Modulates Fibroblast Phenotype
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Fibroblasts seeded on 8-well Lab-Tek II chamber glass slides (Nalgene Nunc International, Naperville, IL, USA) were incubated in serum-free DMEM with 0.1% BSA for 24 h. Fresh media with AdipoRon were added for 24 h. Cells were fixed, permeabilized, and incubated with primary antibodies to α-SMA (Sigma-Aldrich) and type I collagen, both at 1:500 followed by Alexa-fluor-labelled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were identified using 4,6-diamidino-2-phenylindone (DAPI) in ProLong Gold antifade reagent (Invitrogen). Immunofluorescence was evaluated under a Zeiss UV Meta 510 confocal microscope (Carl Zeiss Inc, Jena, Germany).
7
Immunohistochemical Analysis of Mouse and Human Tumors
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Mouse tumours were embedded in optimal cutting temperature (OCT) medium. When relevant, to preserve GFP, tumours were fixed in 4% PFA overnight, and transferred to 30% sucrose for 24 h prior to embedding and freezing in OCT. Formalin-fixed, paraffin-embedded human tumour samples were sectioned and stained with the primary antibodies listed above. For the IHC antibody, binding was detected using the IHC Vector Labs ABC staining kit, before haemotoxylin counterstaining. Stained sections were scanned using the Aperio Scanscope XT (Leica Biosystems, Wetzlar, Germany), and biomarker analysis was conducted using the Aperio image analysis Positive Pixel Count v9 algorithm. For immunofluorescence staining of human tumours, Alexa Fluor-labelled secondary antibodies (Invitrogen, Waltham, MA, USA) were used, nuclei were stained with DAPI, and imaging was carried out using a Zeiss LSM 980 confocal microscope (displayed as a maximum projection of a 6 micron z-stack), using Zeiss imaging software. Human tissue samples were collected following approval from the Austin Health Human Ethics Committee.
8
Fibroblast TMAO Immunofluorescence Assay
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Healthy control and SSc skin fibroblasts at low passage were seeded on 8-well Lab-Tek II chamber glass slides (Nalgene Nunc, Naperville, IL, USA) and incubated in serum-free culture media with 0.1% BSA for 24 h, followed by fresh media with indicated concentrations of TMAO for 48 h. Cells were fixed, permeabilized, and incubated with primary antibodies followed by Alexa-fluor-labelled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were identified using 4,6-diamidino-2-phenylindone (DAPI). Immunofluorescence was evaluated under a Nikon C2 or A1Si confocal microscope.
9
Detecting dsRNA and TLR-3 in Alveolar Macrophages
10
Immunofluorescence Staining of Meiotic Oocytes
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Basically, cultured oocytes were fixed in 2% paraformaldehyde (PFA) in PBS containing 0.1% Triton X-100 for 30 min. Only for microtubule staining, cultured oocytes were preincubated with ice-cold M2 for 10 min and proceeded to fixation as described above. After permeabilization with 0.1% Triton X-100 in PBS overnight at 4°C, oocytes were incubated with primary antibodies overnight at 4°C. Following three washes with PBS containing 0.1% polyvinyl alcohol (PVA), Alexa-Fluor-labelled secondary antibodies (Invitrogen) were used for the detection of signals and DNA was counterstained with 14.3 μM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen). The following primary antibodies were used: anti-REC8 (a kind gift from Jibak Lee, Kobe University, Japan; 1:100), anti-centromere protein (1:100), anti-SGOL2, anti-Meikin (kind gifts from Yoshinori Watanabe, University of Tokyo, Japan; 1:100 and 1:100, respectively), anti-TOPOII (Abcam, #ab109524, 1:100), anti-histone H3 tri-methylated at lysine 9 (H3K9me3; Abcam, #ab8898, 1:250-500) and anti-alpha Tubulin (Sigma, #T9026, 1:250-500). Samples were mounted on a slide with Vectashield (Vector Laboratories), covered with a No.1.5H (170 μm ± 5 μm) coverslip (Marienfield) and were imaged using a confocal laser scanning microscopy.
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