Cells lysates were prepared in 50 mm Tris–HCl, 150 mm NaCl, 1% Nonidet P40 buffer supplemented with protease (Millipore, Livingston, UK), and phosphatase (Millipore) inhibitor cocktails. Proteins were separated on a 10% SDS‐PAGE gel, transferred onto a nitrocellulose membrane and blocked using 5% milk diluted in Tris‐buffered saline with 0.1% tween (TBST) before overnight incubation at 4 °C with a primary antibody (diluted 1:1,000 in 5% bovine serum albumin [BSA]/TBST). Membranes were then washed with TBST, then incubated for 1 h with a species‐appropriate HRP‐conjugated secondary antibody (diluted 1:5,000 in TBST). The horseradish peroxidase (HRP)‐linked secondary antibodies used were goat anti‐mouse (P0447, Dako, Ely, UK) and goat anti‐rabbit (P0448, Dako). Bands were visualised using Immobilon Forte Western HRP Substrate (Millipore) using an Amersham Imager 600 (GE Healthcare, Amersham, UK). Primary antibodies used were mouse anti‐αSMA (1:5000, M0851, Dako), rabbit anti‐fibulin 2 (1:1000, PA521640, Invitrogen), rabbit anti‐ADAMTS14 (1:1000, PA5103578, Invitrogen), and mouse anti‐HSC70 (1:5000, sc7298, Insight Biotechnology, Wembley, UK). Secondary antibodies used were HRP‐conjugated goat anti‐mouse (P0447, Dako) and HRP‐conjugated goat anti‐rabbit (P0448, Dako).
P0448
Manufactured by Agilent Technologies
Sourced in Denmark, United States, United Kingdom, Germany
The P0448 is a laboratory equipment product from Agilent Technologies. It is designed to perform core functions related to analytical testing and measurements. The detailed specifications and intended use of this product are not available for this response.
Automatically generated - may contain errors
Lab products found in correlation
P0447, by Agilent Technologies (46 mentions)
P0260, by Agilent Technologies (17 mentions)
P0449, by Agilent Technologies (7 mentions)
P0161, by Agilent Technologies (7 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
A5441, by Merck Group (5 mentions)
Amersham imager 600, by GE Healthcare (5 mentions)
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
P0160, by Agilent Technologies (3 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Western bright chemiluminescence reagent, by Advansta (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Western bright chemiluminescence reagent, by Cytiva (3 mentions)
Polyclonal goat anti mouse, by Agilent Technologies (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
132 protocols using p0448
1
Western Blot Analysis of Cytoskeletal Proteins
2
Western Blot Analysis of Protein Samples
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Samples for Western blotting were lysed prepared using previously published protocol20 (link) and separated in a 4–20% Mini-PROTEAN® TGXTM Gel (4561093, Bio-Rad, UK). The separated protein in the gel was transferred using the Trans-Blot® TurboTM Transfer system (1704155, Bio-Rad, UK). The membrane was blocked overnight and then incubated with primary antibody for 1 hour (MERTK, Abcam 52968, 1:1000; GAPDH, Everest EB07069, 1:2000; CRALBP, ThermoFisher MA1813, 1:1000), washed and incubated with horseradish peroxidase-conjugated secondary antibodies (P0448, P0449, Dako, UK). The membrane was incubated in Amersham ECL prime (GERPN2232, Sigma) and images collected on the ChemiDoc™ MP System (1708280, Bio-Rad, UK).
3
Cathepsin D and Caspase 3 Immunoblotting Assay
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Protein was isolated in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phosphatase inhibitor cocktail 3 (p0044, Sigma‐Aldrich), 1x cOmplete protease inhibitor cocktail (11 873 580 001, Roche), and 15 mM sodium orthovanadate (S6508, Sigma‐Aldrich). Protein concentration was determined with the DC protein assay kit. Equal amounts of protein were separated by SDS‐PAGE and proteins were transferred to PVDF membrane. For detection of specific proteins, the following antibodies were used: polyclonal anti‐cathepsin D IgG (1:1000; sc‐10 725, Santa Cruz), monoclonal anti‐Caspase 3 (1:1000; 9664, Cell Signalling) and monoclonal anti‐GAPDH IgG (1:30.000; 10R‐G109A, Fitzgerald). After washing, blots were incubated with polyclonal goat anti‐rabbit IgG‐HRP (1:2000; P0448, Dako), and polyclonal rabbit anti‐mouse IgG‐HRP (1:2000; P0260, Dako). Signals were detected visualized with enhanced chemiluminescence (ECL; NEL120001EA, PerkinElmer) and densitometry has been analysed with ImageQuant LAS 4000 (GE Healthcare). Cathepsin D signals were normalized to respective GAPDH levels.
4
Hippocampal Protein Expression Analysis
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Hippocampal regions from WT mice at 3.5 month were dissected, homogenized, and solubilized at 4 °C for 1 h in lysis buffer (1 % CHAPS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4 and protease inhibitors). Primary hippocampal neurons were collected, homogenized and solubilized in the same buffer on DIV14. The total protein lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-STIM1 pAb (1:1000, Cell Signaling, 4916 s), anti-STIM2 pAb (1:1000, AnaSpec, 54681), anti-Phospho-CaMKII (1:2000, Abcam, ab171095), anti-CaMKII (1:1000, Chemicon, MAB8699), anti-PSD95 (1:1000, Cell Signaling, 3450 s), anti-actin clone C4 (1:1000, Millipore, MAB1501) and anti-tubulin (1:1000, DSHB, E7-c). HRP-conjugated anti-rabbit (1:2000, DAKO, P0448) and anti-mouse (1:2000, DAKO, P0447) secondary antibodies. Analysis of the data was performed using Quantity One from BioRad software. The mean density of each band was normalized to actin or tubulin signal in the same sample and averaged. All Western blots replicated 3–7 times.
5
Antibody Characterization for IF and IB
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The following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 (SAB1405371, Sigma Aldrich), GFP (ab290, Abcam) and CENPA (07–574, Millipore; and ab13939, Abcam). For immunoblotting, the following primary antibodies were used: SA1 (ab4457, Abcam), SA2 (A300-158a, Bethyl Laboratories), SMC1 (A300-055A, Bethyl Laboratories), SCC1 (05-908, Millipore), WAPL (A-7, sc-365189, Santa Cruz), Sororin (ab192237, Abcam), HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin (T5168, Sigma Aldrich). All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1 (A300-055A, Bethyl Laboratories) or IgG (2729 S, Cell Signaling) per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse (A-11001, Life Technology), Alexa Fluor 568 goat anti-mouse (A-11004, Life Technology), Alexa Fluor 488 goat anti-rabbit (A-11008, Life Technology) and Alexa Fluor 568 goat anti-rabbit (A-11011, Life Technology). For western blots, we used the following secondary antibodies: anti-goat-PO (P0449, DAKO), anti-rabbit-PO (P0448, DAKO) and anti-mouse-PO (P0447, DAKO).
6
Antibodies for Surfactant Protein Analysis
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The antibodies used were as follows: mouse anti-human surfactant protein A (SP-A) monoclonal antibody (MAB3270; Chemicon-Millipore, Hampshire, UK); mouse anti-human surfactant protein B (SP-B) monoclonal antibody (MAB3276; Chemicon-Millipore, Billerica, USA); rabbit anti-human SP-C (proSP-C) monoclonal antibody (AB3786; Chemicon-Millipore, Billerica, USA); rabbit anti-human SP-D monoclonal antibody (BM4083; Acris, San Diego, USA). Mouse*/Rabbit** anti-human Vimentin antibody (*M7020; DAKO—Glostrup, Denmark/** ab45939; Abcam) was used to characterize cells of Sertoli and Leydig. Secondary antibodies: anti-rabbit*/anti-mouse** IgG, respectively, conjugated to horseradish peroxidase (*P0448; Dako, Glostrup, Denmark/**80807; Dianova, Hamburg, Germany), anti-rabbit*/anti-mouse** IgG, respectively, conjugated to Alexa-Fluor 488 and Rhodamin-Red (*31665, **A11001, Thermo Fisher Scientific/life technologies). All antibodies mentioned were used for Western blot analysis as well as for immunohistochemical investigation as specified by the manufacturer. The antibodies used are highly specific, showing no cross-reactivity with other cellular proteins, and have been used successfully in previous experiments [11 (link),12 (link),16 (link)].
7
CD8+ T Cell Quantification in Murine Tumors
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C57BL/6 mice bearing MC38 tumors (average volume of 80 mm3) were treated with muC5H9v2 mAb, CX-630 Pb-Tx, or isotype control (5 mg/kg) on days 0, 3, and 7. On day 8, tumors were excised, fixed in formalin, and embedded in paraffin blocks. CD8 immunohistochemical analysis was performed by Cureline, Inc. Briefly, 5 μm sections were cut, deparaffinized, and rehydrated followed by antigen retrieval using an EDTA buffer. Sections were washed and incubated in a blocking solution. Primary staining was performed with a 200-fold dilution of rabbit anti-CD8 antibody (Abcam, catalog #ab217344) or isotype control (Abcam, catalog #ab172730) in staining buffer (PBS with 0.5% Tx-100 and 1% FBS) for 1 hour at room temperature. After several washes, tissue sections were stained with an anti-rabbit HRP-conjugated secondary antibody for 20 minutes at room temperature (Dako, catalog #P0448). After a final wash step, the slides were incubated with a 3,3′-diaminobenzidine chromogen for 10 minutes at room temperature. After rinsing, slides were counterstained with hematoxylin, dehydrated, cleared, and mounted. Brightfield whole-tissue scans where performed on the slides using the Vectra Polaris Imaging System (Akoya Biosciences) at a 20× magnification. Snapshots images were captured digitally from the scan at 10× magnification using Phenochart software version 1.0.
8
Western Blot Analysis of Lipid Metabolism Proteins
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Total cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) as well as Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich). Samples were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were hybridized with rabbit anti-adipose triglyceride lipase (ATGL; 2138, 1:1,000; Cell Signaling Technology), rabbit anti-phospho-hormone-sensitive lipase (HSL; Ser563; 4139, 1:1,000; Cell Signaling Technology), rabbit anti-HSL (4107, 1:1,000; Cell Signaling Technology), mouse anti-LXRα (ab41902, 1:1,000; Abcam), rabbit anti-LXRβ (ab28479, 1:500; Abcam), and mouse anti-GAPDH (MAB374, 1:80,000; Millipore) antibodies at 4°C overnight to detect expression, followed by 1-h incubation at room temperature with goat anti-mouse (sc-2005, 1:4,000; Santa Cruz Biotechnology) and goat anti-rabbit (P0448, 1:2,000; Dako) peroxidase-conjugated antibodies. Proteins were detected by chemiluminescence (Millipore).
9
Protein Extraction and Western Blot Analysis
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Cells were pelleted by centrifugation at 200 X g for 5 minutes and resuspended in 200 μl RIPA buffer with a protease inhibitor cocktail (Sigma-Aldrich Company Ltd., Germany). Lysates were incubated on ice for 30 minutes before centrifuging at 10,000 X g for 20 minutes to remove cell debris. Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL). Thirty to 50 μg of protein lysates were resolved by SDS-polyacrylamide gel (12%). The gels were electroblotted onto nitrocellulose membranes (Hybond-P, Amersham, GE Healthcare). Antibody staining was performed with a chemiluminescence detection system (Supersignal; Pierce). PARP (14-6666;eBioscience, Cheshire, UK), SHH (ab53281; Abcam, Cambridge, UK), hyperacetylated histone H3 (#9715; Cell Signalling, Leiden, NL), H3K9Ac (#9649; Cell Signalling, Leiden, NL) and acetylated α-tubulin (#3971; Cell Signalling, Leiden, NL) monoclonal antibodies were used in conjunction with a horseradish peroxidase (HRP)-conjugated sheep anti-mouse or anti-rabbit secondary antibody (P0447 and P0448; Dako, Denmark). Equal loading was assessed using a β-actin mouse monoclonal primary antibody (Sigma, Poole, Dorset, UK).
10
Western Blot Analysis of Bpifa1 Protein
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Bulla fluids were sampled by pipetting as previously described48 (link), these were then mixed with an equal volume of 2XSDS lysis buffer. Samples were denatured at 95 °C for 5 minutes and resolved on a 12% polyacrylamide gel, transferred to a PVDF membrane using a semi dry blotting system (Biorad Trans-blot turbo) then probed with rabbit anti-Bpifa1 primary antibody (1:20031 (link)), overnight at 4 °C. The primary antibody was detected using a polyclonal goat anti-rabbit secondary antibody (Dako P0448) conjugated with HRP (1:2000) and bound antibody was visualised using the EZ-Chemiluminescence detection system (Geneflow, Cat No- 30500500B) and manual development. Images were scanned as a whole with no modification.
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