Brains were fixed in formalin (4% buffered formalin), and embedded in paraffin. Coronal sections were stained by H&E (haematoxylin and eosin) or immunostained for human nucleolin (hNCL, 1:200, 4 °C, overnight; ab13541, Abcam, Cambridge, UK; does not react with mouse) to visualize the human GBM cells using a heat antigen retrieval procedure as previously described58 (link).
Ab13541
Manufactured by Abcam
Sourced in United Kingdom
Ab13541 is a laboratory instrument used for performing spectrophotometric analysis. It measures the absorbance or transmittance of light passing through a sample, which can be used to quantify the concentration of substances in the sample.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using ab13541
1
Histological Analysis of Human GBM Cells
2
Nucleolin Immunohistochemistry in FFPE Tissue
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Tissue samples were formalin fixed and paraffin embedded. Serial sections deparaffinized and rehydrated through an alcohol gradient to water, and antigen retrieval in citrate buffer pH 6.0 was used for the human nucleolin antibody at 5.0 g/mL (ab13541) (Abcam, Cambridge, MA). Endogenous peroxide activity and nonspecific binding were blocked with 3%(vol/vol) peroxide and 2% (vol/vol) normal horse serum. Primary antibody and anti-mouse ImmPRESS-HRP secondary antibody were incubated for 1 hr and visualized using 3,3′-diaminobenzidine (Vectorlabs, Burlingame, CA). Normal horse serum or monoclonal IgM was used in control sections.
3
Xenograft Tumor Characterization Workflow
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At final sacrifice, the fresh mouse brain was cut using a brain mold. The central coronal brain slice (~ 5 mm) encompassing the injection-site was frozen in OCT (Tissue-Tek), and the rest was formalin fixed and paraffin embedded for histology. Serial frozen sections were cut on a cryostat from the central slice of the mouse brains. The first and last sections were used to determine tumor location and estimate tumor cell content, based on hematoxylin and eosin (H&E) staining and nuclear immune-positivity for human-specific NCL (ab13541, Abcam, Cambridge, UK) as previously described [29 (link)]. Xenografts were macro-dissected, guided by H&E and NCL staining, and collected from the injected and contralateral side separately. In absence of apparent spread to the contralateral side, the tissue of the symmetric area on the contralateral side was collected. Immunohistochemistry was performed for EGFR (E30), Ki67 (MIB1), GFAP (G-A-5), and TP53 (DO-7) (platform Ventana, Roche).
4
Immunostaining of Mouse Brain Proteins
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Mouse brains were fixed in 4% paraformaldehyde for 16 h, embedded in paraffin, and sectioned at a thickness of 5 μ5. The sections were de-paraffinized in xylene and re-hydrated, followed by antigen retrieval by microwaving in 0.01 M citrate buffer (pH 6.0) at 120 °C for 15 min. After permeation with PBS containing 0.5% Triton X-100, the sections were incubated with blocking solution (10% normal donkey serum in PBS) for 60 min at room temperature, following by incubation for 24 hours at 25 °C with mouse anti-Htt (1:100, MAB5374, Sigma Aldrich), mouse anti-Atxn1 (1:100, MAB5374, Sigma Aldrich), mouse anti-fibrillarin (1:100, #ab4566, Abcam), rabbit anti-nol10 (1:5000, #ab181161, Abcam), and mouse anti-nucleolin (1:5000, #ab13541, Abcam) primary antibodies. After washing three times in PBS at room temperature, the sections were incubated at room temperature for 1 h with Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000, A21206, Molecular Probes) and Alexa Fluor 555-conjugated anti-mouse IgG (1:1000, #A31570, Molecular Probes) secondary antibodies.
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