FFPE Sections (4-μm thick) were mounted and routinely stained with H&E for histopathological examination (Supplementary Fig. S1a ). Multiplex immunofluorescence was applied to identify the expression patterns of key transcriptomic regulators of SCLC, including ASCL1 (Abcam, ab211327), NEUROD1 (Abcam, ab60704) and POU2F3 (Novus Biologicals, NBP1–83966). Multiplex immunofluorescence staining was performed using a PANO 7-plex IHC kit (Panovue, Cat# 0004100100), as previously described.65 In brief, the FFPE sections were subjected to deparaffinization, rehydration, and antigen retrieval according to the protocol supplied by the manufacturer. After blocking, the sections were incubated with a primary antibody and then a secondary antibody (polymer HRP-anti-mouse/Rabbit IgG). Other primary antibodies were sequentially applied by repeating the previous procedures. Nuclei were stained with DAPI (Sigma-Aldrich, D9542) after all the human antigens had been labeled. Multispectral images were obtained by scanning the stained slides with the Mantra System (PerkinElmer, Waltham, Massachusetts, US) and analyzed using inForm image analysis software (PerkinElmer, Waltham, Massachusetts, US) (Fig. 4c ).
Ab211327
Manufactured by Abcam
Ab211327 is a monoclonal antibody that targets a specific protein. It is designed for use in various laboratory applications, including Western blotting, immunohistochemistry, and ELISA. The product details and intended use are provided by the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop, by Roche (2 mentions)
Ab32072, by Abcam (2 mentions)
D9542, by Merck Group (1 mentions)
Nbp1 83966, by Novus Biologicals (1 mentions)
Mantra system, by PerkinElmer (1 mentions)
Inform image analysis software, by PerkinElmer (1 mentions)
Pano 7 plex ihc kit, by Panovue (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Target retrieval solution s1699, by Agilent Technologies (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ecl prime western blot detection kit, by GE Healthcare (1 mentions)
Ab8926, by Abcam (1 mentions)
Ab52627, by Abcam (1 mentions)
Na934, by GE Healthcare (1 mentions)
Subcellular fractionation kit, by Thermo Fisher Scientific (1 mentions)
Na931, by GE Healthcare (1 mentions)
Ab1791, by Abcam (1 mentions)
9 protocols using ab211327
1
Multiplex Immunofluorescence Analysis of SCLC Markers
2
Immunoblotting for Transcription Regulators
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For immunoblotting, cell protein extracts were prepared following standard procedures in RIPA buffer in the presence of protease inhibitors (Complete, Roche Diagnostics) and phosphatase inhibitors (PhosStop, Roche Diagnostics). After quantitation with the Micro‐BCA Protein Assay Kit (Thermo Fisher Scientific), 25 µg of protein was separated by SDS–PAGE and transferred to PVDF membranes (Immobilon‐P, Millipore). After using appropriated primary and secondary antibodies, blots were developed by a peroxidase reaction using ECL detection system (Amersham‐G.E. Healthcare). Antibodies used were Recombinant Anti‐NeuroD1 antibody [EPR4008] (ab109224; 1:1,000 dilution), Anti‐MASH1/Achaete‐scute homolog 1 (Abcam ab211327; 1:1,000 dilution), Mouse Anti‐RNAPII 7c2 (IGBMC Antibody facility; 1:1,000 dilution), and Anti‐Phospho‐Serine 2 RNAPII (Abcam ab5095; 1:1,000 dilution). Anti α‐Tubulin (SIGMA T5168) (Novus Biologicals hVIN‐1 NB600‐1293; 1:1,000 dilution) was used as loading control. All the blots are representative of at least three independent experiments.
3
Immunoperoxidase Analysis of Lung Tissue
4
Western Blot Analysis of Cell Lysates
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Cells were lysed on ice with RIPA buffer (Sigma) supplemented with 1X protease inhibitor cocktail (Roche). Proteins were separated on SDS-PAGE and immmunoblotted with rabbit anti-ASCL1 (1:1000, ab211327, Abcam), mouse anti-Rb (1:1000, 9309, Cell Signalling), rabbit anti CDKN1C (1:1000, 2557, Cell Signalling) and mouse anti-tubulin (1:1000, 66301, Proteintech). Secondary antibodies were horseradish peroxidase (HRP) conjugated (Amersham) and were detected with ECL Prime Western Blot Detection Kit (GE Healthcare).
5
Immunohistochemical Analysis of Tumor Samples
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Tumor tissues were fixed in 4% formaldehyde and embedded in paraffin. Sections (5 mm) were deparaffinized and heat antigen-retrieved in citrate buffer (pH 6.4). Primary antibody staining was performed by incubating overnight at 4 °C, and endogenous peroxidase (HRP) activity was blocked by treating the sections with 3% hydrogen peroxide in methanol. Indirect IHC was performed with antispecies-specific biotinylated secondary antibodies, followed by avidin-horseradish peroxidase and developed using DAB color substrates (Nichirei Biosciences Inc., Tokyo, Japan). The sections were counterstained with haematoxylin. The following primary antibodies were used: Anti-MASH1/Achaete-scute homolog 1 (Abcam, ab211327, 1:100); anti-NOTCH1 (Abcam, ab52627, 1:100); anti-NOTCH2 (Abcam, ab8926, 1:100).
6
Subcellular Protein Fractionation and Analysis
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Protein was extracted by resuspending cells in RIPA buffer and incubating for 20 min. Debris was removed by centrifuging at 16,000xg for 10 min. Protein lysates were separated on a BisTris gel (Invitrogen), transferred to a nitrocellulose membrane and blocked using 5% milk. Primary antibodies were diluted in 1% milk and incubation took place overnight at 4 °C. Primary antibodies were as follows; ASCL1 (ab211327, abcam), CMYC (ab32072, abcam), GATA3 (D13C9, Cell Signalling Technologies), NMYC (9405, Cell Signalling Technologies), PHOX2B (sc-376997, Santa Cruz), α-Tubulin (66031, ProteinTech), GAPDH (60004, ProteinTech) and Histone H3 (ab1791, abcam). Secondary antibodies were diluted in TBST and incubation took place at room temperature for 1 hour; anti-Mouse (NA931, GE Healthcare) anti-Rabbit (NA934, GE Healthcare)
To analyse protein content in different parts of the cell, the Subcellular Fractionation Kit was used and followed according to manufacturers instructions (78840, Thermo Fisher). Briefly, a fresh cell pellet is resuspended in specific buffers to sequentially extract the cytoplasmic, membrane, soluble nuclear and chromatin bound proteins. Protein isolated was subject to western blot analysis (above).
To analyse protein content in different parts of the cell, the Subcellular Fractionation Kit was used and followed according to manufacturers instructions (78840, Thermo Fisher). Briefly, a fresh cell pellet is resuspended in specific buffers to sequentially extract the cytoplasmic, membrane, soluble nuclear and chromatin bound proteins. Protein isolated was subject to western blot analysis (above).
7
ASCL1 Phosphorylation Detection Assay
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To detect phosphorylation of ASCL1, proteins were treated with 400 units of Lambda Protein Phosphatase (New England BioLabs) for 30 min at 30 °C. ASCL1 phosphorylation was visualised on 8% acrylamide gels polymerized with 20 μM “Phos-tag” (WAKO) and 40 μM MnCl2. After electrophoresis, “Phos-tag” gels were washed three times for 10 min with transfer buffer (14.4 g Glycine; 3 g Trizma Base; 800 mL H2O; 200 mL MeOH; final pH 8.3) plus 10 mM EDTA, and then once more with normal transfer buffer. Proteins were transferred to nitrocellulose membranes and immunoblotted with rabbit anti-ASCL1 (1:1000, ab211327, Abcam) and mouse anti-tubulin (1:1000, Sigma).
8
Western Blot Analysis of Transcription Factors
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Cells were washed with phosphate‐buffed saline and then lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail tablets (cOmplete; Roche Diagnostics) and phosphatase inhibitor tablets (PhosSTOP; Roche Diagnostics). Protein contents were quantified using XL‐Bradford (SDS‐PAGE Adapted) (Integrale). Lysates (10 μg) were resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and electrotransferred onto polyvinylidene difluoride membranes (Immobilon; Merck Millipore). After blocking membranes for 90 min at room temperature, they were incubated overnight at 4°C with one of the following primary antibodies (1:1000): rabbit monoclonal anti‐ASCL1 (ab211327; Abcam), rabbit monoclonal anti‐NEUROD1 (ab109224; Abcam), mouse monoclonal anti‐POU2F3 (sc‐293 402; Santa Cruz Biotechnology), and rabbit monoclonal anti‐YAP1 (14 074; Cell Signaling Technology). Hsp90 levels were used as a control for protein loading. After incubation with primary antibodies, membranes were incubated with a secondary antibody for 1 h at room temperature. Horseradish peroxidase‐linked anti‐rabbit (Cytiva) and anti‐mouse (Cytiva) antibodies were used as secondary antibodies. Proteins were detected by enhanced chemiluminescence (LAS‐4000; Cytiva).
9
LuCaP UW Tissue Microarray Protocol
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The LuCaP UW TMA 103 was constructed from subcutaneous PDX tumors. It contains 39 LuCaP PDX models; 3 tumors per PDX model, and 3 punches per PDX model (together 9 cores per model). The antibodies used for IHC were as follows: HOXB 13 (Cell Signaling Technologies, cat no. #90944, 1:50, antigen retrieval pH6), SYP (Santa Cruz sc-17750, 1:100 antigen retrieval pH6), AR (BioGenex MU256-0717 1:100 Antigen retrieval pH9), ASCL1 (ABCAM ab-211327, 1:100, antigen retrieval pH6). The scoring of each TMA was performed as previously published (29 (link)).
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