We assessed CXCL13 by RNA ISH (RNAscope 2.5 HD Reagent kit [RED]; Advanced Cell Diagnostics). FFPE tissue sections were deparaffinized in xylene and subsequently dehydrated in an ethanol series. Tissue sections were incubated in target retrieval reagent at 100°C for 15 minutes and were then treated with protease at 40°C for 30 minutes. Hybridization with Hs-CXCL13 (for human) or Mm-Cxcl13 (for mouse) probes at 40°C for 2 hours, and the amplifier and visualization (Fast RED) procedures, were performed in accordance with the manufacturer’s instructions. For multiplex detection using FFPE, an RNAscope Fluorescent Multiplex Reagent kit v2 (Advanced Cell Diagnostics) was used. Double staining was performed for CXCL13 and CD8, CXCL13 and CD4, and CXCL13 and CD21 (CR2). The target probes were Hs-CXCL13 (C1), Hs-CD4 (C2), Hs-CD8A (C2), and Hs-CR2 targeting 269-1303 of NM_001006658.3 (C2). CXCL13 was detected with Opal 690 (1:1000 dilution, PerkinElmer), and CD4, CD8, and CR2 were detected with Opal 570 (1:1500 dilution, PerkinElmer). Fluorescence images were captured using a fluorescence microscope BZ-X800E (KEYENCE), and the colocalization of CXCL13 with various immune cells was quantified using BZ-H4C/hybrid cell count software (KEYENCE).
Bz h4c hybrid cell count software
Manufactured by Keyence
Sourced in Japan
The BZ-H4C/hybrid cell count software is a laboratory equipment product that provides automated cell counting functionality. It is designed to accurately and efficiently count cells in a variety of samples. The core function of this product is to perform cell enumeration tasks.
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2 protocols using bz h4c hybrid cell count software
1
RNA ISH Detection of CXCL13 in Immune Cells
2
Multiplex Analysis of CXCL13 Expression
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We assessed CXCL13 by RNA in situ hybridization (ISH) (RNAscope ® 2.5 HD Reagent kit (RED), Advanced Cell Diagnostics, Hayward, CA, USA). FFPE tissue sections were deparaffinized in xylene and subsequently dehydrated in an ethanol series. Tissue sections were incubated in target retrieval reagent at 100°C for 15 minutes, and then treated with protease at 40°C for 30 minutes. Hybridization with Hs-CXCL13 (for human) or Mm-Cxcl13 (for mouse) probes at 40°C for 2 hours, and the amplifier and visualization (Fast RED) procedures were performed in accordance with the manufacturer's instructions. For multiplex detection using FFPE, an RNAscope ® Fluorescent Multiplex Reagent kit v2 (Advanced Cell Diagnostics) was used. Double staining was performed for CXCL13 and CD8, CXCL13 and CD4, and CXCL13 and CD21(CR2). The target probes and reagents used were listed in Supplemental Table S2. CXCL13 was detected with Opal 690, and CD4, CD8, and CR2 with Opal 570. Fluorescence images were captured using a fluorescence microscope BZ-X800E (KEYENCE, Osaka, Japan), and the colocalization of CXCL13 with various immune cells was quantified using BZ-H4C/hybrid cell count software (KEYENCE).
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