For immunofluorescence (IF) staining of FGFR3, 5μm FFPE sections were subjected to antigen retrieval with citrate buffer for 8 min. Primary antibodies were: FGFR3-N (1:400, sc-13121, Santa Cruz Biotechnology), FGFR3-C (1:2000, sc-123, Santa Cruz Biotechnology), TACC3-N (1:600, ab134153, Abcam), and TACC3-C (1:300, NBP1-01032, Novus Biological). Secondary biotinylated antibodies were used at 1:50,000 followed by streptavidin and TSA Cy3-conjugated. Nuclei were counterstained with DAPI. For immunohistochemical analysis (IHC) of FGFR3 expression, antigen retrieval was performed for 12 min and FGFR-3 antibody (sc-13121, Santa Cruz Biotechnology) was diluted 1:500. Biotinylated anti-mouse antibody (1:30,000) and streptavidin were added before incubation with the chromogen. Nuclei were counterstaining with hematoxylin.
Sc 13121
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-13121 is a laboratory product offered by Santa Cruz Biotechnology. It serves as a general-purpose instrument for scientific research applications. The core function of this product is to facilitate the measurement and analysis of various biological and chemical samples.
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3 protocols using sc 13121
1
Immunostaining Protocols for FGFR3 and TACC3
2
Immunohistochemical Analysis of FGFR3 Expression
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TMA slides (4 μm) were stained with a mouse monoclonal antibody raised against the N-terminal region of FGFR3 (specifically against amino acids 15-124 of FGFR3 of human origin, SC-13121, Santa Cruz Biotechnology, Dallas, TX; dilution 1:200), using the Ventana Benchmark Ultra automated staining instrument (Ventana Medical Systems, Tucson, AZ), according to manufacturer instructions. Expression of FGFR3 was semiquantitatively scored by 1 neuropathologist (W.H.), based on staining intensity: 0 (negative, N), 1+ (low, L), 2+ (high, H), as described previously by Theelen et al.21 (link) The highest score of the 3 TMA tissue cores from each sample was used to select cases for further analysis, to compensate for intratumoral heterogeneity. Of the samples that showed at least 1 TMA core with positive FGFR3 staining, corresponding whole-tumor FFPE slides were stained as well.
3
Immunohistochemical Analysis of FGFR3 Expression
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Immunohistochemistry for FGFR3 was performed on sections cut from the paraffin blocks with a commercially available rabbit monoclonal Anti-FGFR3 antibody (sc-13121, Santa Cruz, USA). Slides were sectioned at 4μm onto positively charged slides (Superfrost Plus, Menzel-Glaser, Germany) and the slides were stained on an automated platform the (Dako Autostainer Link 48). Heat-induced antigen retrieval was used for 30 min at 97°C in the high-PH EnVision™ FLEX Target Retrieval Solution, and the primary antibody was used at a dilution of 1 in 100. The detailed histopathological assessment was done regarding confirmation of diagnosis and grading of malignant cases.
Immunohistochemical assessment was done according to [9 (link)]. Cells with membrane cytoplasmic staining were considered positive: The intensity and extent of cells stained positively for FGFR3 was evaluated by assessing 10 high-power microscopic fields by two independent pathologists (OH &TA): *Score 0: All tumor cells are negative; *Score +1: Weak but detectable positivity in some or all cells; *Score +2: moderate but extensive positivity; and *Score +3: strong positivity (regardless of extent).
Immunohistochemical assessment was done according to [9 (link)]. Cells with membrane cytoplasmic staining were considered positive: The intensity and extent of cells stained positively for FGFR3 was evaluated by assessing 10 high-power microscopic fields by two independent pathologists (OH &TA): *Score 0: All tumor cells are negative; *Score +1: Weak but detectable positivity in some or all cells; *Score +2: moderate but extensive positivity; and *Score +3: strong positivity (regardless of extent).
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