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4 protocols using donkey anti mouse dylight 549

1

Multiprotein Immunofluorescence Imaging

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Primary antibodies from commercial sources were mouse anti-giantin, -VDAC (Abcam, Cambridge, MA), -ATP synthase α (Mitosciences, Abcam) rabbit anti-lamin A/C, -p38 MAPK (Cell Signaling Technologies, Danvers, MA); rabbit anti-nSMase3, -nSMase2, mouse anti-annexin 2 (ECM Biosciences, Versailles, KY); and rabbit anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies were as follows: donkey anti-mouse Alexa 680 (Molecular Probes-Invitrogen); goat anti-rabbit IRDye 800CW (LI-COR); donkey anti-rabbit DyLight 649; and donkey anti-mouse DyLight 549 (Jackson Immunoresearch, West Grove, PA).
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2

Antibodies and Plasmids for CSPP Research

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Antibodies used in this study were as follows: anti-CSPP-L (Proteintech Europe, Manchester, United Kingdom; used for IF at 1 : 1000), anti-CSPP/CSPP-L (described in (Patzke et al, 2010 (link)); IF 1 : 200), anti-cytokeratin-8 (AbCam, Cambridge, UK; IF 1 : 500), anti-smooth muscle actin (Abcam; IF 1 : 100), anti-Cyclin A (Santa Cruz Biotechnology, Santa Crux, CA, USA; IF 1 : 1000), anti-γ-Tubulin (Sigma, St Louis, MO, USA; IF 1 : 500), and anti-Pericentrin (Abcam; IF 1 : 400). Secondary fluorochrome conjugated antibodies (Donkey anti-rabbit DyLight488; Donkey anti-mouse DyLight549; IF 1 : 1000) were purchased from Jackson Immuno Research (West Grove, PA, USA). shRNA and GFP co-expression plasmids used for CSPP1 knockdown were obtained from SA Biosciences (part of Qiagen N.V., Venlo, Netherlands): set KH18087G with the following target sequences: shRNA_01: 5′-gcacgaattcagcaggagtat-3′, shRNA_02: 5′-tccttcagttgacagcatcat-3′, shRNA_03: 5′-ggtgccaaagttgacttagat-3′, shRNA_04: 5′-ggaggtgaagatcgagaactt-3′, and shRNA_control: 5′-ggaatctcattcgatgcatac-3′. Tagged CSPP and CSPP-L full-length and truncation protein expression plasmids pCSPPmyc (derived from AJ583433) and pCSPP-Lmyc (derived from AM156947), pCSPP(498-876-eGFP) were described earlier (Patzke et al, 2006 (link)).
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3

Immunocytochemistry of Neural Markers

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The following primary antibodies were used: anti-MBP (rat, 1:800, Chemicon),
anti-neurofilament (mouse, 1:800, SMI31, Covance), anti-Olig1 (rabbit, 1:1000,
Abcam), anti-p75NTR (rabbit, 1:500, Promega), anti-S100 (rabbit,
1:500, Dako).
The following secondary antibodies were used: donkey-anti-rabbit AlexaFluor488,
donkey-anti-mouse DyLight549, donkey-anti-rat AlexaFluor647 (all 1:500, Jackson
ImmunoResearch Laboratories), DAPI (4′,6-diamidino-2-phenylindole)
(1.25 μg/ml; Molecular Probes) was used as cellular counter
stain.
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4

Perfusion and Immunohistochemistry of Rodent Brains

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Adult animals were anesthetized with a ketamine (250 mg/kg) and xylazine (25 mg/kg) solution (i.p.) and transcardially perfused with 4% paraformaldehyde in PBS (1 ml/g). E18.5 embryos were excised after deep anesthesia of pregnant mothers; embryos' brains were extracted and immersed in fixative. After anesthesia in isoflurane, P0–P2 brains were extracted and immersed in fixative, whereas P3–P8 pups were perfused with paraformaldehyde before brain extraction. Brains were postfixed overnight, washed in PBS, then placed in 30% sucrose in PBS before freezing in liquid nitrogen and kept at –20°C for long‐term storage.
Coronal or horizontal brain sections were prepared and processed for immunohistochemistry as described previously (Picardo et al., 2011). Various primary antibodies were used (Table 2). Secondary antibodies (dilution 1:500) used were donkey anti‐chicken DyLight 488, donkey anti‐rabbit DyLight 549, donkey anti‐guinea pig Cy3, donkey anti‐mouse DyLight 549, and donkey anti‐goat DyLight 649 (all from Jackson Immunoresearch, West Grove, PA).
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