For the immunofluorescence in Figure 3 D, twelve gravid adult hermaphrodites were dissected in 5 μl of M9 buffer on a poly-L-lysine coated slide. A 13 mm2 round coverslip was placed over the M9, and slides were placed into a container with liquid nitrogen. After flicking away the coverslip, samples were fixed for 20 minutes in −20°C methanol. Samples were re-hydrated in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4), twice for 5 minutes, blocked with AbDil (PBS, 2% BSA, 0.1% Triton X-100) in a humid chamber for 30 minutes at room temperature, and incubated in AbDil with rabbit anti-SMA-1 PH antibody (1:1000) and anti-α-tubulin antibody (1:1000, DM1-α, Sigma), overnight at 4°C. After washing four times for 5 minutes in PBS, samples were incubated in AbDil with Alexa Fluor 488 anti-rabbit IgG (1:300; Jackson ImmunoResearch) and Alexa Fluor 594 goat anti-mouse IgG (1:300; Jackson ImmunoResearch) for 1 hour at room temperature. Samples were washed four times for 5 minutes in PBS and mounted in Prolong Gold (Invitrogen).
Alexa fluor 488 anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in Panama, United States
Alexa Fluor 488 anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Anti c myc 9e10, by Santa Cruz Biotechnology (1 mentions)
Cryostat, by Leica (1 mentions)
Anti ki67 antibody ab15580, by Abcam (1 mentions)
A1 microscope, by Nikon (1 mentions)
Tunel assay, by Promega (1 mentions)
Alexafluor 647 anti guinea pig, by Jackson ImmunoResearch (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
Ab2785, by Abcam (1 mentions)
Imagej software, by National Instruments (1 mentions)
Alexa fluor 647 anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Protein block serum free reagent, by Agilent Technologies (1 mentions)
Anti dystrophin, by Abcam (1 mentions)
Alexa fluor 594 anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
8 protocols using alexa fluor 488 anti rabbit igg
1
Immunofluorescence Staining of Hermaphrodite Samples
2
Immunofluorescence Staining of c-Myc and FUS
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Mice were deeply anesthetized with the MMB combination anesthetic (i.e., 0.3 mg/kg of medetomidine hydrochloride, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol tartrate) and then perfused intracardially with freshly prepared 2% paraformaldehyde in phosphate buffer for 10 min. Brains were dissected and soaked in the same fixative for 2 h on ice. For cryoprotection, the tissue pieces were placed into 20% sucrose solution for 4 h and 25% sucrose solution overnight, after which they were quickly frozen using liquid nitrogen and stored at −80 °C. Serial 7-µm-thick sections were cut using a cryostat (Leica Biosystems, Wetzlar, Germany). The sections were blocked with 1% BSA/10% normal donkey serum/0.5%Triton X-100/M.O.M. Blocking Reagent in PBS for 1 h at room temperature, followed by incubation with the primary anti-c-Myc 9E10 (1:100; sc-42; Santa Cruz Biotechnology, Dallas, TX) and FUS polyclonal (1:100; PA5-23696; Thermo Fischer Scientific, Waltham, MA) antibodies overnight. The sections were then incubated with the secondary antibodies Cy3 anti-mouse IgG (1:100; 715-166-151; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 anti-rabbit IgG (1:100; 711-546-152; Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The embedded samples were imaged with a laser scanning confocal microscope (LSM700; Carl Zeiss, Oberkochen, Germany).
3
Quantifying Cell Proliferation and Apoptosis
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Frozen sections were fixed in 4% paraformaldehyde in PBS. For Ki67 staining to image cell proliferation, the sections were blocked with blocking solution (0.3% Triton X, 5% horse sera, and 1% goat sera in PBS). Anti-Ki67 antibody (ab15580) (Abcam, Cambridge, MA) was applied to the blocking solution followed by the addition of Alexa Fluor® 488 anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). To evaluate cell apoptosis, a TUNEL assay was performed, according to the manufacturer's protocol (Promega Corporation, Madison, WI). All images were acquired using a Nikon A1 microscope (Nikon Inc., Melville, NY).
4
Quantification of Munc13-1 Presynaptic Expression
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Autaptic neurons at DIV 15-20 were fixed for 10 min in 4% PFA, permeabilized with 0.1% PBS-Tween-20 solution and blocked with 5% normal donkey serum. Rabbit anti-VGLUT1 (SYSY 135302) and Guinea-pig anti-Munc13-1 (SYSY 126104) primary antibodies were used for coimmunostaining to detect Munc13-1 presynaptic expression. The primary antibodies were labeled with AlexaFluor-488 anti-rabbit IgG, as well as AlexaFluor-647 anti-guinea pig both in donkey serum (Jackson ImmunoResearch Laboratories), respectively. Single neurons on the astrocytic micro-islands were imaged using a Nikon scanning confocal microscopy A1Rsi with a × 60 oil immersion objective. The z-series images at 0.3 µm depth were obtained at equal exposure times with 1024 × 1024 pixels resolution and at the pixel size of 0.2 µm. The analysis was performed blind in ImageJ software by drawing ROIs of 50 synapses per neuron. ROIs were defined by staining for the SV marker VGLUT1. Munc13-1 fluorescence intensity for each synapse was divided to the corresponding fluorescence intensity of VGLUT1; ∼30 neurons per group were analyzed in three independent cultures, and the data were normalized to the control of each experiment.
5
Colon Cell Identification by FISH and Immunostaining
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Colon sections were fixed in methanol-Carnoy’s solution and embedded in paraffin. The colon slices were hybridized with a TAMURA-labeled Eub338 probe (key resources table ) in a hybridization solution including 5 M NaCl, 1 M Tris-HCL, formamide, and nuclease-free water at 50°C overnight after deparaffinizing. After washing, the colon slices were blocked with 10% donkey serum (Sigma Aldrich) for 15 min and treated with a rabbit muc2 polyclonal antibody (1:100; Novus Biologicals) in the dark at room temperature overnight. After washing, colon slices were visualized using Alexa Fluor 488 anti-rabbit IgG (1:500; Jackson ImmunoResearch) and Hoechst 33342 (1:200; Thermo Fisher Scientific) for 3 h at room temperature in the dark. Colon slices were then enclosed in ProLong Gold Antifade Mountant (Invitrogen).
6
Galectin-3 Immunofluorescence in Cell Lines and Tissues
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A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody (#ab2785, Abcam, USA) in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., USA) in 2% BSA at room temperature for 1 hour.
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).
7
Dystrophin and Collagen III Analysis in Muscle
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Fresh muscle samples were rapidly frozen in isopentane cooled with liquid nitrogen. Different mice from those used for the force measurement were used for immunohistological staining. Using a cryostat, 7 μm-thick frozen sections were prepared. Fresh frozen sections were fixed with acetone for 5 min at −20 °C and blocked with a protein block serum-free reagent (Agilent, Santa Clara, CA, USA) for 15 min. The specimens were incubated with primary antibodies at 4 °C overnight, followed by secondary staining. The primary and secondary antibodies used were anti-dystrophin (1:800; Abcam, Cambridge, UK, Cat# ab15277), anti-collagen III (1:100; Abcam, #ab7778), Alexa Fluor 488 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA, #711-545-152), and Alexa Fluor 594 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, #711-585-152). Stained samples were mounted with SlowFade Diamond anti-fade reagent (Thermo Fisher, Waltham, MA, USA, S36972). Immunofluorescent images were obtained using an inverted fluorescence microscope DMI6000B (Leica, Wetzlar, Germany), BZ-X710 (Keyence, Osaka, Japan), and a confocal laser scanning microscope system TCS SP8 (Leica). Quantitative analyses of dystrophin and collagen III staining were performed using the Hybrid Cell Count application (Keyence).
8
Visualizing Drosophila Gut Morphology
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Fly guts were collected as described for gut measurements. Guts were fixed in 4% paraformaldehyde in PBS and Alexa-Fluor-488–phalloidin (Life Technologies A12379; 1:500) was used to visualize actin filaments. For phospho-histone H3, guts were fixed and stained as described previously (Micchelli and Perrimon, 2006 (link)) using an antibody that recognizes phospho-serine 10 (Sigma #H0412; 1:200). Secondary antibodies used were Alexa-Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch 111-545-144; 1:250) and guts were whole-mounted using Vectashield (Vector Labs).
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