At the experimental end-point animals were euthanized and tissues were fresh frozen in liquid nitrogen or fixed in 4% paraformaldehyde in PBS overnight. To review histology, slides were stained by haematoxylin and eosin (H&E). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using standard protocols. Specificity of immunostaining was assessed by incubation in the absence of primary antibody. We used the following primary antibodies: RFP for Strawberry detection (ab34771, 1:400; Abcam), Aquaporin 1 (NB-600–749, 1:500; Novus Biologicals), THP (AF5175, 1:100; R&D), Aquaporin 2 (ab105171, 1:1000; Abcam), Nephrin (AF3159, 1:100; R&D), CD34 (ab8158, 1:50; Abcam), CD73 (AF4488, 1:100; R&D), GFP (ab290, 1:1000; Abcam), HIF2a (NB100-132, 1:150; Novus Biologicals), CA9 (sc-25600, 1:200; Santa Cruz), turbo-RFP (AB234, 1:500; Evrogen) and pS6 (2211, 1:200; Cell Signalling). Secondary antibodies used were conjugated to HRP (IHC) or Alexa-fluor® fluorochromes (IF). Fluorescent images were obtained by confocal laser-scanning microscopy (Leica TCS SP5).
Ab34771
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab34771 is a laboratory equipment product. It is designed for general laboratory use. The core function of this product is to facilitate specific tasks or processes within a laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Ab13970, by Abcam (4 mentions)
Polymer refine kit, by Leica Biosystems (2 mentions)
Aperio at2, by Leica Biosystems (2 mentions)
Fv1000d, by Olympus (2 mentions)
Ab8158, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Nb100 132, by Novus Biologicals (1 mentions)
Nb 600 749, by Novus Biologicals (1 mentions)
Af3159, by R&D Systems (1 mentions)
Af4488, by R&D Systems (1 mentions)
Sc 25600, by Santa Cruz Biotechnology (1 mentions)
Ab234, by Evrogen (1 mentions)
Tcs sp5, by Leica camera (1 mentions)
Ab7463, by Abcam (1 mentions)
Axio observer z1 fluorescent microscope, by Zeiss (1 mentions)
Ab78286, by Abcam (1 mentions)
Alexa fluor 488 conjugated, by Thermo Fisher Scientific (1 mentions)
Ab34151, by Abcam (1 mentions)
Ab7349, by Merck Group (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
19 protocols using ab34771
1
Histological and Molecular Characterization
2
Immunofluorescence Staining of Tissue Sections
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Gels were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned. Sections were prepared for staining by deparaffinizing in a xylene substitute, rehydration, and heat-mediated antigen retrieval using pH 9 tris-EDTA with 0.05% tween 20. Sections were blocked in 10% goat serum and incubated with primary antibodies in a humidified chamber at 4 °C overnight. Secondary antibodies were added for 1 h at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Antibodies were used at the following concentrations: anti-green fluorescent protein (anti-GFP) rabbit IgG Alexa Fluor 488 conjugated (1:75; Invitrogen A21311, Invitrogen, Carlsbad, CA, USA), rabbit polyclonal antibody to GJB1 [Cx32] (1:25; HPA010663, Sigma-Aldrich, St. Louis, MO, USA), biotinylated rabbit polyclonal antibody to red fluorescent protein (RFP) (1:500; Abcam, Cambridge, UK, ab34771), rabbit anti-laminin 1 + 2 (1:100; Abcam, ab7463), and mouse anti-laminin 5 antibody (1:100; Abcam, ab78286), Alexa Fluor 488 and 568 conjugated goat secondary antibodies (1:1000; Thermo Fisher Scientific) or avidin Alexa Fluor 488 (Thermo Fisher Scientific A21370). All sections were counterstained with DAPI and imaged by using a Zeiss axio-observer Z1 fluorescent microscope.
3
Quantifying Cells in Tissue Samples
4
Multimodal Immunolabeling of Oligodendrocytes
5
Protein Extraction and Western Blot Analysis
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Cells were extracted using M-Per reagent (Thermo Scientific) with Complete Mini Protease inhibitors (Roche), and protein concentration determined using the Bradford assay. Extracts were run on an SDS PAGE gel (NuPAGE, Invitrogen), transferred to a nitrocellulose membrane and luciferase-YFP detected using polyclonal goat anti-luciferase antibody (g475A, Promega), at 1 in 25,000 dilution with incubation for 1 h at room temperature. For α-tubulin staining a monoclonal mouse anti-α-tubulin (T1799, Sigma-Aldrich) was used at 1 in 1,000 dilution with overnight incubation at 4 °C. For mStrawberry staining, rabbit polyclonal anti-RFP (ab34771, AbCam) was used at 1 in 10,000 dilution with overnight incubation at 4 °C. Horseradish peroxidase conjugated anti-rabbit (111-035-003), anti-goat (705-035-003-JIR), and anti-mouse (115-035-006-JIR) antibodies (Jackson ImmunoResearch, Suffolk, UK) were used as the secondary antibodies at 1 in 10,000 dilution with an incubation time of 45 min at room temperature. All antibodies were diluted with 5 % w/v milk powder and 0.1 % Tween.
6
Western Blotting of Protein Samples
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Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (GE Healthcare). After being blocked in 1× PBST buffer containing 5% skimmed milk, the membrane was incubated with the selected primary antibody using a 1,000-fold dilution overnight at 4 °C, washed three times with 1× PBST (10 min each) and incubated with the selected secondary antibody conjugated with horseradish peroxidase using a 3,000-fold dilution for 1 h at room temperature. After three washes with 1× PBST (10 min each), the film was illuminated and photographed with ImageQuant800 (GE Healthcare). AMERSHAM ImageQuot 800 software (v.1.2.0) was used for western blotting analysis; GeneSys software (v.1.3.8.0) was used for gel imaging. If the primary antibodies were already conjugated with horseradish peroxidase, there was no need to incubate the membrane with secondary antibodies. The following antibodies were used: anti-GFP (GF28R, 1:1,000 dilution, Thermo Scientific), anti-RFP (ab34771, 1:2,500 dilution, Abcam), anti-FLAG (F3165, 1:3,000 dilution, Sigma), anti-HA (3F10, 1:3,000 dilution, Sigma), anti-mouse (sc-516102, 1:1,000 dilution, Santa Cruz) and anti-rabbit (ab205718, 1:5,000 dilution, Abcam).
7
Evaluating Lung Tumors in Rodents
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At the experimental end-point and immediately after a routine in vivo bioluminescence image, tumour-bearing or control animals were euthanized; the lungs were then quickly removed, inflated with PBS and imaged ex vivo with an IVIS 200 series camera. The lungs were then fixed in 4% PFA for 24 h, embedded in paraffin, and five 4 µm-thick sections were cut at 100 µm steps, with one slide stained with haematoxylin and eosin (H&E) at each step, resulting in 8–12 step sections covering the whole three dimensions for each pair of lungs. These step sections were analysed for tumour formation, with counting of tumours (which were often present in several adjacent step sections). Tumours were assessed for the presence of invasion to distinguish adenocarcinomas from adenomas, and the adenomas were graded into those showing low-grade or high-grade dysplasia, using established criteria 20 (link).
Immunohistochemistry (IHC) was performed on unstained sections following microwave-based antigen retrieval in citrate buffer and incubation with an anti-RFP primary antibody (ab34771; Abcam) at 1:400 dilution at 4°C overnight. The primary antibody was then detected by incubation with a biotinylated secondary antibody (diluted 1:200) and horseradish peroxidase (Vectastain ABC Kit, PK4005, Vector Laboratories). The slides were counterstained with haematoxylin.
Immunohistochemistry (IHC) was performed on unstained sections following microwave-based antigen retrieval in citrate buffer and incubation with an anti-RFP primary antibody (ab34771; Abcam) at 1:400 dilution at 4°C overnight. The primary antibody was then detected by incubation with a biotinylated secondary antibody (diluted 1:200) and horseradish peroxidase (Vectastain ABC Kit, PK4005, Vector Laboratories). The slides were counterstained with haematoxylin.
8
Pairwise Immunoprecipitation Assay in Nicotiana benthamiana
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For the pairwise immunoprecipitation assays, two 1 cm diameter leaf discs from two N. benthamiana leaves transiently expressing the tagged proteins were homogenised in extraction buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% v/v Triton X‐100, 1× protease inhibitor cocktail, 1 mM DTT). Homogenates were spun at 20 000 g , 10 min, and proteins were collected in the supernatant (Input sample). Immunoprecipitation was performed on the input sample using the μMACS GFP Isolation Kit (Miltenyi Biotec, Woking, UK) or the RFP‐Trap Magnetic Particles (Chromotek, Planegg, Germany) and μMACS Columns (Miltenyi Biotec). For immunoblotting, antibodies were used in the following concentrations: 1 : 5000 anti‐GFP (TP401; Torrey Pines, Biolabs, Secaucus, NJ, USA), 1 : 2000 anti‐RFP (ab34771; Abcam, Cambridge, UK) and 1 : 5000 anti‐HA (ab9110; Abcam). Bands were detected using the anti‐rabbit IgG (whole molecule)‐Peroxidase (A0545; Sigma) at 1 : 20 000 dilution and the SuperSignal West Femto Trial Kit (Thermo Scientific).
9
Quantifying Cells in Tissue Samples
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Sections of formalin-fixed paraffin-embedded tissue (10 μm for xenografts and 5μm for brain tissue) were stained with H&E or with an anti-RFP rabbit polyclonal antibody that crossreacts with mStrawberry (1:100 dilution) (Abcam ab34771) and detected using Leica’s Polymer Refine Kit in combination with the Bond automated system (Leica Biosystems DS9800). Slides were scanned at x20 magnification with a resolution of 0.5 μm per pixel on an Aperio AT2 (Leica Biosystems). Images were analyzed using the Aperio Imagescope Nuclear version 9 algorithm to quantify the proportion of cells with positive nuclear staining.
10
Immunoprecipitation of GFP/RFP-tagged Proteins
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Fifteen to twenty seedlings were grown in wells of a 6-well plate for 2 weeks in liquid MS media with gentle agitation (6 wells were used per treatment). The MS media was replaced the night before treatment. Seedlings were treated with 1 μM elf18/SCOOP12 for 10 min before flash-freezing. Tissue was ground and proteins extracted in 1:1 (v/v) powdered tissue:extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (P9599, Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4 and 1% IGEPAL) for 30 min at 4 °C. Cell debris was removed via centrifugation. For immunoprecipitation, GFP-TRAP/RFP-TRAP AGAROSE BEADS (ChromoTek) were incubated with extracts for 3 h at 4 °C and washed three times in wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (P9599, Sigma Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, and 0.1% IGEPAL) before adding 2× Laemmli sample buffer and incubating for 10 min at 95 °C. Detection was carried out by SDS-PAGE and western blots using α-BAK119 (link) α-RFP (ab34771, Abcam) and α-GFP-HRP (sc-9996, Santa Cruz) antibodies. α-rabbit-HRP (A-0545, Merck) was used to detect α-BAK1 and α-RFP.
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