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4 protocols using ab32072

1

Immunohistochemical Analysis of Mouse Tissues

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Tissues were formalin-fixed overnight and paraffin-embedded by routine procedures. Haematoxylin and eosin (HE) and immunohistochemical stainings were performed by standard protocols. The following primary rabbit antibodies were used for immunohistochemistry: anti-Myc (Abcam ab32072), anti-CD3 (Thermo Scientific, RM-9107), anti-F4/80 (abD serotec, MCA497), anti cleaved caspase-3 (cell signaling, #9661), anti Ki67 Abcam (ab15580) and anti-CD31 (AbCam ab28364). All slides were digitally processed using the Aperio ScanScope (Aperio, Vista, CA, USA) and captured using ImageScope software version 12.0.0 (Aperio) or quPath version 0.3.061 (link).
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2

Tissue Analysis of Immune Markers

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Tissues were formalin-fixed overnight and paraffin-embedded by routine procedures.
Haematoxylin and eosin (HE) and immunohistochemical stainings were performed by standard protocols. The following primary rabbit antibodies were used for immunohistochemistry: anti-Myc (Abcam ab32072), anti-CD3 (Thermo Scientific, RM-9107), anti-F4/80 (abD serotec, MCA497) and anti-CD31 (AbCam ab28364). All slides were digitally processed using the Aperio ScanScope (Aperio, Vista, CA, USA) and captured using ImageScope software version 12.0.0 (Aperio).
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3

Multi-immunostaining of Tissue Sections

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Tissue samples were fixed in 4% paraformaldehyde (EMS) at room temperature for 45 minutes, followed by an overnight incubation in 30% weight/volume sucrose solution (Sigma-Aldrich). Samples were then embedded in optimal cutting temperature (Tissue-Tek) and frozen on dry ice. Staining was performed on 10-μm sections by first blocking with 5% donkey serum and 0.1% Tween-20 for 1 hour, followed by overnight incubation with primary antibody diluted in blocking buffer in a humidified chamber. Sections were washed three times in PBS containing 0.1% Tween-20. For immunofluorescence staining, slides were then incubated with DAPI (Life Technologies, 1:1,000) and Alexa fluorophore–conjugated antibodies (Jackson ImmunoResearch). For IHC, slides were first incubated with biotinylated secondary antibodies (Jackson ImmunoResearch) and developed using the ABC HRP and DAB kits per manufacturer protocols (Vectorlabs). Primary antibodies used were as follows: rat anti–Ki-67 (eBioscience, 14–5698–82), rabbit anti–c-Myc (Y69; Abcam, Ab32072), rabbit anti-CD3 (Invitrogen, PA1–29547), rabbit anti-F4/80 (Novus, NBP2–12506), rat antineutrophil (Abcam, NIMP_R14), anti-CD206 (R&D Systems, AF2535), and rabbit anti-Arg1 (Cell Signaling Technology, 93668).
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4

Western Blot Analysis of Cellular Proteins

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Cells expressing individual sgRNAs were induced in 1 μg/mL
doxycycline for 2 to 5 days before lysis in Laemmli buffer and incubation at
95°C for 5 min. For mitotic samples, cells were harvested by mitotic
shake off and, when necessary, after an overnight 10 μM STLC
incubation. Samples were separated by SDS-PAGE and semi-dry transferred to
nitrocellulose. Membranes were blocked for 30 min in blocking buffer (5% BSA
for H2A.X; for all others, milk in TBS with 0.1% Tween-20) before incubation
with primary antibodies: anti-phospho-H2A.X (Ser139, Millipore clone JBW301;
1:1000), anti c-Myc (Abcam, ab32072; 1:1000), anti-CENP-A (Clone
3–19, Invitrogen; 1:2000), or
anti-“Bonsai”/NDC8093 (link) (0.5 μg/mL). This was
followed by HRP-conjugated secondary antibody (Kindle Biosciences)
incubation at 1:1000 dilution. To detect GAPDH as a loading control,
HRP-conjugated antibody (Abcam, ab185059) was applied at 1:20,000 dilution.
Membranes were imaged with a KwikQuant Imager (Kindle Biosciences) and
quantified using Image Studio software (LI-COR).
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