Hk080 9k

Manufactured by BioGenex

The HK080–9K is a compact and automated tissue staining system designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. It features a compact footprint and supports a range of staining protocols.

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Lab products found in correlation

Dylight 488 anti rabbit igg green, by Vector Laboratories (1 mentions) Dylight 549 anti mouse igg red, by Vector Laboratories (1 mentions) Mouse anti human cd24 antibody, by Abcam (1 mentions)

3 protocols using hk080 9k

Multiplex IHC for Immune Profiling of Tumor Biopsies

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Paired tumor biopsies were obtained at baseline and week 7 (after at least two doses of GVAX + IPI) in 6 patients on Arm A. A pathologist selected samples containing >30% tumor cellularity for 10-plex multiplex IHC. Multiplex IHC was conducted on 4 micron thick formalin fixed paraffin embedded (FFPE) tissue sections mounted on slides as previously described(18 (link)). Iterative staining was achieved by washing and antibody stripping in heated citrate (HK080–9K BioGenex, Fremont, CA). Samples were restained sequentially with antibodies indicated (Supplementary Table S2, Supplementary Figure S2.A).
Whole-slide scans were obtained after each stain on Hamamatsu Scanner at 20x magnification. Digital image analysis consisted of three steps: 1) image coregistration using Cell Profiler 2 (Version 2.2.0, Broad Institute) with “coregistration 12 markers” pipeline, 2) visualization in ImageJ Version 2 (Java 1.8.0_172 (64-bit), NIH), and 3) quantitative image analysis of staining intensity was performed in three areas per FFPE tissue (≥6.25mm²) using Cell Profiler 2 with “cytometry 12 markers” pipeline and FlowFCS Express 6 Image Cytometry Version 6.06.022 (De Novo Software) (Supplementary Figure S2). Images were merged in Halo (version 2.0.1145.31, Indica labs) to identify immune subsets according to lineage markers described (Supplementary Table S3).

Wu A.A., Bever K.M., Ho W.J., Fertig E.J., Niu N., Zheng L., Parkinson R.M., Durham J.N., Onners B., Ferguson A.K., Wilt C., Ko A.H., Wang-Gillam A., Laheru D.A., Anders R.A., Thompson E.D., Sugar E.A., Jaffee E.M, & Le D.T. (2020). A Phase 2 Study of Allogeneic GM-CSF Transfected Pancreatic Tumor Vaccine (GVAX) with Ipilimumab as Maintenance Treatment for Metastatic Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(19), 5129-5139.

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Immunohistochemical Identification of Macrophages and T-Cells

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Macrophages/monocytes were revealed using an antigen retrieval procedure (#HK080‐9K; BioGenex Laboratories, San Ramon, CA), in which the coronal floating sections were heated at 80°C for 35 min in 10 mmol/L citrate solution (pH 6.0) and cooled to room temperature. Sections were then rinsed in distilled water, washed in 0.1 mol/L PB for 10 min and incubated in a blocking buffer (5% donkey serum) in 0.1 mol/L PB at room temperature for 1 h. They were then incubated overnight at 4°C with the blocking buffer containing a primary antibody: mouse monoclonal anti‐macrophages/monocytes (clone ED‐1; Millipore, Billerica, MA; MAB1435; 1/300) or mouse monoclonal anti‐T‐cell (clone 15‐6A1; Santa Cruz Biotechnology, Dallas, TX; sc‐52711; 1/1000), then washed in PB three times and exposed to Alexa‐Fluor 488‐linked donkey anti‐mouse (#A21202; Life Technologies, Carlsbad, CA) immunoglobulins diluted 1:200 with the blocking solution for 2 h at room temperature.
For the detection of rat IgG, coronal sections were incubated in the same blocking buffer for 1 h and then with Alexa Fluor 488 donkey anti‐rat IgG (H+L) antibody (#A21208; Life Technologies) diluted 1:200 with the blocking solution for 2 h at room temperature.

Niquet J., Gezalian M., Baldwin R, & Wasterlain C.G. (2015). Neuroprotective effects of deep hypothermia in refractory status epilepticus. Annals of Clinical and Translational Neurology, 2(12), 1105-1115.

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Immunofluorescence Staining of CD90 and CD24

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Protein expression was determined by immunofluoresence (IF) staining [7 (link)]. Briefly, the tissue specimens were placed in xylene twice for 20 minutes and hydrated in 100% ethanol, 95% ethanol, 75% ethanol, 5 minutes each and water for 3 minutes. Antigen retrieval was completed by boiling the sections in solution (BioGenex HK080-9K) for 15 minutes. Subsequently, these were blocked with 2% BSA in PBS containing 0.05% Tween (PBST) for 1 hour at room temperature and incubated with rabbit anti-human CD90 antibody (1:100, Abcam, Cambridge, MA) or mouse anti-human CD24 antibody (1:150, Abcam, Cambridge, MA) at 4°C overnight. The secondary antibodies of DyLight 488 anti-rabbit IgG (green) or DyLight 549 anti-mouse IgG (red, Vector Laboratories, Burlingame, CA) were used at 1:150 dilution and incubated in a dark place for 1 hour at room temperature. CD90 staining was visualized in green and CD24 in red. Staining with DAPI (blue, 1:4000 dilution) presented nuclei visualization. The sections were dehydrated in alcohol and xylene, then cover-slipped with TM mounting medium (Sigma, USA).

Pei X., Zhu J., Yang R., Tan Z., An M., Shi J, & Lubman D.M. (2016). CD90 and CD24 Co-Expression Is Associated with Pancreatic Intraepithelial Neoplasias. PLoS ONE, 11(6), e0158021.

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