Eyes were fixed in 4.0% paraformaldehyde for 2 h at room temperature, transferred into 30% sucrose for cryopreservation and embedded in OCT (Tissue Tek). Tissue was stored at -80o C until used. Cryostat sections were cut in the transverse plane at 10 microns and stored at -20o C until IF protocol. Slides were permeabilized with 0.5% Triton-X-100 in PBS (PBS-T) for 30 min at room temperature. Slides were washed 3x PBS for 5 min each. Following permeabilization, slides were blocked with 4.0% BSA for 1 h at room temperature. Slides were washed 3x PBS for 5 min each. Slides were incubated for 24 h at 4 C with primary antibodies diluted in 1.0% BSA and PBS-T in a humidity chamber. Slides were then washed 4x PBS for 10 min each. Slides were incubated with secondary antibodies diluted in 1.0% BSA in PBS-T for 2 h at room temperature in humidity chamber at room temperature then washed 3x PBS for 10 min each, then a final wash in PBS with Hoechst (1:2,000) for 10 min. Slides were imaged on Leica SP8 at 63x magnification. Antibodies and dyes: CTCF (Santa Cruz sc-271,514, 1:100), Alexa Flour 488 (Jackson 115-547-185, 1:250) and Hoechst 33,342 (Fisher H3570, 1:2,000).
Alexa flour 488
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488 is a fluorescent dye produced by Jackson ImmunoResearch. It is a bright, green-fluorescent dye that can be used for a variety of applications, including immunofluorescence, flow cytometry, and other fluorescence-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Histochoice, by Merck Group (2 mentions)
H3570, by Thermo Fisher Scientific (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Anti mre11, by Cell Signaling Technology (1 mentions)
Mirin, by Merck Group (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Merck Group (1 mentions)
Alexaflour 546, by Jackson ImmunoResearch (1 mentions)
Lipofectamine 2000 3000, by Thermo Fisher Scientific (1 mentions)
Anti fade gold, by Thermo Fisher Scientific (1 mentions)
Sch900776, by Selleck Chemicals (1 mentions)
Crispr cas9, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Cryostar nx50 cryostat, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Neun antibody, by Merck Group (1 mentions)
Cyp1b1 antibody, by Abcam (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Cd11c, by Proteintech (1 mentions)
13 protocols using alexa flour 488
1
Immunofluorescence Imaging of CTCF in Cryosections
2
Immunofluorescence Analysis of DNA Damage Markers
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Antibodies against TOP1, IκBα, phospho‐ATM, γH2AX, DNA. FLAG, β‐actin, Anti‐IKKγ/NEMO TDP1 (#SAB1411073), and H2AX were purchased from Sigma (St. Louis, MO). Antibodies against RPA2 and phospho‐RPA2 were from Bethyl Laboratories (Montgomery, TX). Antibodies against p65, WRN (#SC5629), ATM (#SC23921), CHK1 (#SC8404), Lamin B (#SC6216), and CRISPR‐Cas9 double nickase plasmid (control and WRN) were from Santa Cruz biotechnology (Santa Cruz, CA). Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK). Anti‐BrdU (#347580), anti‐PAR from BD Biosciences (San Jose, CA). Anti MRE11 (#4847) from Cell Signaling Technology (Massachusetts, USA). AlexaFlour‐546 and AlexaFlour‐488 tagged secondary antibodies from Jackson ImmunoResearch, (PA, USA). Lipofectamine 2000/3000, Alexa Flour‐488/555/595 (#A21123/#A31570), prolong anti‐fade Gold was obtained from (Life Technologies, Carlsbad, CA). SCH900776 (CHK1 inhibitor) was purchased from Selleckchem (Houston, USA). Talazoparib (BMN673; PARP inhibitor) was procured from ApexBio, USA. All other reagents like Ro106‐9920, mirin (MRE11 inhibitor), camptothecin, EdU, etc., were obtained from Sigma Chemicals (St. Louis, MO), unless mentioned in the respective places.
3
Spinal Cord Tissue Preparation and Immunostaining
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Animals were pericardially perfused on a down draft perfusion table with heparinized phosphate buffered saline followed by 4% paraformaldehyde. Spinal cord tissues were extracted and post-fixed overnight, then cryoprotected using 10%, 20%, and 30% sucrose solutions. Spinal cords were embedded in Tissue-Tek OCT compound (Sakura, Holland) and snap frozen in liquid nitrogen. Samples were sectioned longitudinally with 20 µm thickness on a Cryostar NX50 cryostat (ThermoFisher, MA) and mounted on slides for staining. Slides were blocked with 10% Donkey serum (Millipore Sigma, MA) and 0.5% Triton X-100 (Millipore Sigma, MA) in phosphate buffered saline for 1 h at room temperature. Longitudinal 20 µm sections of spinal cord tissue were stained for rabbit GFAP (AB72650, Abcam, United Kingdom) at a concentration of 1:500 for 2 h, washed 3× in phosphate buffered saline for 7 min, and counterstained with donkey anti-rabbit AlexaFlour 488 (AB2313584, Jackson ImmunoResearch, PA) for 1 h. Sections were again rinsed 3× with phosphate buffered saline for 7 min each and coverslips were attached using 80uL of Fluoromount-G (Southern Biotech, AL). Slides were imaged using LAS X software suite on a Leica DMI8 inverted confocal microscope (Leica Microsystems, Germany).
4
Immunohistochemical Analysis of Brain Sections
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Immunohistochemistry was performed on 14 µm coronal brain sections. Slides were placed in room temperature for 30 min, and thereafter placed in 4% buffered formaldehyde for 10 min. Slides were then rinsed in 0.01 M PBS and incubated in a humid chamber at 5 °C for 21 h with either CYP1B1 antibody (Abcam, Product ID: ab185954) diluted 1:2000 or CPR antibody (Abcam, Product ID: ab180597) diluted 1:100 together with NeuN antibody (Millipore, Catalog No: MAB377) diluted 1:1000, 5% donkey serum and 95% primary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton + 5% bovine serum albumin). Thereafter, slides were rinsed in 0.01 M PBS and incubated in room temperature for 30 min with secondary antibodies Cy3 (Jackson ImmunoResearch, Product ID: 711165152) and Alexa Flour 488 (Jackson ImmunoResearch, Product ID: 715545150), both diluted 1:400 with secondary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton). Thereafter slides were rinsed in 0.01 M PBS and mounted with Mowiol.
5
Immunofluorescence Staining of FFPE Tumor Samples
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Immunofluorescence staining was performed on formalin fixed paraffin embedded (FFPE) tumor tissue sections and adherent cells. The tumor tissues were fixed in 10% formalin, embedded in paraffin, and serially sectioned for 3 µm thick. The following primary antibodies were used: CD11c (Proteintech, 17342–1-AP, 1:100), TSHα (Proteintech, 25014–1-AP, 1:100), TSHβ (SantaCruz, D-6, 1:50), TSHR (SantaCruz, C-10, 1:50), PD-L1 (Abcam, EPR19759, 1:250), total c-JUN (CST, 9165, 1:400), phosphorylated c-JUN (CST, 3270, 1:100). Double stainings of TSHR with PD-L1 were performed manually. Primary antibodies were detected with whole IgG or IgG F(Ab’)2 fragments conjugated to Alexa Flour 488 (711-546-152, Jackson Immuno Research) or streptavidin-conjugated Cy3 (016-160-084, Jackson Immuno Research). 4′,6-diamidino-2-phenylindole (DAPI) was used for visualization of cell nuclei. Sigma-Aldrich). For immunofluorescence multiplex staining, we followed the staining method for the following markers: CD11c with fluorescein isothiocyanate FITC (1:50); TSHα with fluorescein Cy3 (1:50); TSHβ with fluorescein Cy5 (1:50) and nuclei visualized with DAPI (1:2,000). A Nikon Ti-E microscope was used for all imaging. Image analysis was performed using NIS software modules (Nikon, V.4).
6
Immunohistochemical Analysis of LC3B
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Paraffin-embedded tumor sections were deparaffinized with HistoChoice (Sigma-Aldrich, H2779) and rehydrated with graded alcohol treatments. Antigen retrieval was carried out by microwave treatment for 22 min in citrate buffer (pH 6.0). Sections were blocked in 10% normal donkey serum in PBS and incubated overnight with primary antibody LC3B (rabbit polyclonal, Novus NB600-1384, 1:100) followed by secondary antibody (Alexa Flour 488, Jackson ImmunoResearch 711-545-152, 1:300) and DAPI incubation for nuclei visualization. Average green intensity per image was calculated with ImageJ software, based on three different areas of similar size chosen in a blinded manner.
7
Recombinant TGF-β1 Autophagy Regulation
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Recombinant human TGF-β1 (580704) was obtained from Biolegend (San Diego, CA). CQ (C6628), 3-MA (M9281) and Baf a1 (B1793) were all purchased from Sigma-Aldrich (St. Louis, MO). The anti-LC3B antibody (L7543) was obtained from Sigma-Aldrich. Oil Red O (O9755) was purchased from Sigma-Aldrich. BODIPY 558/564 Red C12 (D3835) and BODIPY 493/503 (D3922) were both obtained from Invitrogen. DAPI (D9542) was obtained from Sigma-Aldrich, and Alexa Flour 488 or 594-conjugated anti-mouse or anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The anti-Beclin-1 antibody (NBP1-00085) was purchased from Novus Biologicals (Littleton, CO). Anti-LAMP1 (ab24170), anti-SQSTM1 (ab56416) and anti- α-SMA (ab124964) antibodies were all obtained from Abcam (Cambridge, MA). Anti-cleaved caspase-3 antibody (#9664) was obtained from Cell signaling Technology (Danvers, MA), and anti-GAPDH antibody (sc-365062) was purchased from Santa Cruz (Santa Cruz, CA).
8
Immunohistochemical Characterization of Xenopus Embryos
9
Immunohistochemistry of Brain Sections
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The paraffin-embedded brain was cut into sections (thickness 3 μm). First, brain sections were deparaffinized in xylene and rehydrated by ethanol (2 × xylene, 100% ethanol, 100% ethanol, 95% ethanol, 75% ethanol, each 5 min). Then, sections were repaired using 800 mL citrate buffer antigen for 20min and washed 3 × 5 min with 0.1 M phosphate-buffered saline (PBS). After being blocked with blocking solution for 120 min, tissues were incubated with the primary antibodies overnight at 4°C: anti-GFAP (1:1500, Sigma, Millipore-MAB360) and anti-IBA-1 (1:400, WAKO, 019-19741). After washing three times (10 min per time) with 0.1 M PBS, slides were incubated with secondary antibodies for 60 min: anti-mouse CY3 (1:1000, Jackson Immunoresearch, United States) for GFAP detection and anti-rabbit Alexa flour 488 (1:1000, Jackson Immunoresearch, United States) for IBA-1 detection. Subsequently, sections were re-washed in 0.1 M PBS for three times (5 min per time). Finally, all sections were counterstained with DAPI (1:100, Solarbio) and mounted using glycerin.
10
Quantifying c-Fos Expression in Hindbrain
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The hindbrain was isolated and sectioned into 100 µm pieces using a vibratome (Leica, VT1000S), and sections were placed in a 24-well plate and stained for c-Fos. Sections were incubated with primary c-Fos antibodies (Rabbit,1:250, Cat. # AB190289, Abcam or Guinea pig, 1:500, Cat# 226,308, SYSY) in PBS with 0.1% Triton X-100 (PBST) overnight at 4 °C. Following incubation, sections were washed 3–4 times with PBST for 5 min and incubated with fluorophore conjugated secondary antibodies (Alexa Flour 594, 1:500, Cat. # 150,080, Abcam or Alexa Flour 488,1:500, Cat# 106–545-003, Jackson ImmunoResearch Labs) in PBST for one hour and wash repeated with PBS. Following addition of ProLong Gold Antifade Mountant (Cat. # P36930, ThermoFischer), tissue sections were observed under a light microscope (ZEISS AxioZoom V16 Fluorescent Microscope) at 63X and 150X magnification and the right and left NTS analyzed for c-Fos positive cells with the help of brain atlas. Analysis was performed for each area by manual counting of c-Fos active cells in two microscopic fields (right NTS and left NTS) per brain slice at 63X from two individuals blinded to experimental conditions and average taken.
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