Expression of Musashi-1 was evaluated semiquantitatively by a 0–3 scale (0 = no staining; 1 = weak; 2 = moderate; 3 = intense) in osteocytes, osteoblasts, osteoclasts, epithelial cells in the Schneiderian membrane, mesenchymal cells, fibroblasts, smooth muscle cells, endothelial cells and adipocytes. To do so, rehydrated samples were also termically treated in a pre-treatment module (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing a 1 mM EDTA buffer (pH = 8) at 95 °C for 20 minutes. Primary polyclonal antibody against Musashi-1 was then applied and incubated at 1:100 dilution for 16 h at 4 °C. Vimentin (clone V9) (1:100 dilution) was used as a positive control. A non-immunospecific IgG was used as negative control. All antibodies were obtained from Master Diagnóstica (Granada, Spain). The immunostaining was developed in an automatic immunostainer (Autostainer480S, Thermo Fisher Scientific Inc.) using a peroxidase-conjugated micropolymer and diaminobenzidine (Master Diagnóstica).
Autostainer480s
Manufactured by Thermo Fisher Scientific
Sourced in Sweden
The Autostainer480S is a compact, automated slide staining system designed for clinical and research laboratories. It performs routine staining procedures efficiently and consistently. The Autostainer480S can handle a variety of slide formats and staining protocols, enabling standardized and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Pre treatment module, by Thermo Fisher Scientific (2 mentions)
Citrate buffer ph 6, by Thermo Fisher Scientific (1 mentions)
Primary antibody enhancer, by Thermo Fisher Scientific (1 mentions)
Dab plus substrate system, by Thermo Fisher Scientific (1 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Vslide, by MetaSystems (1 mentions)
4 protocols using autostainer480s
1
Immunohistochemical Analysis of Musashi-1 Expression
2
Immunohistochemical Analysis of Bone Cell Markers
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A group of sections was stained using immunohistochemical techniques in order to visualize the expression, location, and number of positive cells per mm2 for CD44 (osteocytes), CD56 (osteoblasts), TRAP (osteoclasts), Musashi‐1 (mesenchymal stromal cells), CD45 (all leukocytes), CD68 (monocytes/macrophages) and CD34 (endothelial cells around vessels). Additionally, osteopontin was detected as a marker of osteoconduction. Briefly, after deparaffinizing and rehydrating the slides, they were treated in a pre‐treatment thermal PT module (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a 1mM EDTA buffer (pH 8) for 20 min at 95°C. Then, primary antibodies were applied at a pre‐determined concentration for 1 h at room temperature. The staining was then visualized in an automatic immunostainer (Autostainer480S, Thermo Fisher Scientific Inc.) using a peroxidase‐conjugated micropolymer and diaminobenzidine. All antibodies were purchased from Vitro‐Master Diagnóstica (Granada, Spain) and used following the manufacturer instructions.
3
Immunohistochemical Evaluation of SATB1 and SATB2
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The specificity of SATB1 and SATB2 antibodies was further evaluated in immunohistochemical experiments.
Tissue sections (4 μm) were cut from TMAs containing 18 normal (fallopian tube, cervix, endometrium, placenta, testis, prostate, liver, pancreas, rectum, colon, stomach, duodenum, small intestine, cerebellum, cerebral cortex, skin, skeletal muscle, and tonsil) and 7 cancer (prostate, colorectal, ventricular, renal, liver, lung, and breast) tissues. Prior to immunostaining, the sections were baked at 50 °C overnight and deparaffinized in xylene and graded ethanol. Antigen retrieval was then performed using citrate buffer pH 6 (ThermoFisher Scientific, Waltham, MA, USA) in decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Sections were stained with anti-SATB1rabbit monoclonal antibody (Clone EPR3895, Epitomics, Burlingame, CA, USA) diluted 1:100 or mouse monoclonal antibody against SATB2 (AMAb90679, CL0320, Atlas Antibodies, Stockholm, Sweden) diluted 1:1,000 in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA) using a commercial kit (UltraVision LP HRP polymer®, Primary Antibody Enhancer, Ultra V Block and DAB plus substrate system®, ThermoFisher Scientific, Waltham, MA, USA). Slides were counterstained with hematoxylin and mounted using Pertex.
Slides were examined, and images were taken using an automated system (VSlide, Metasystems).
Tissue sections (4 μm) were cut from TMAs containing 18 normal (fallopian tube, cervix, endometrium, placenta, testis, prostate, liver, pancreas, rectum, colon, stomach, duodenum, small intestine, cerebellum, cerebral cortex, skin, skeletal muscle, and tonsil) and 7 cancer (prostate, colorectal, ventricular, renal, liver, lung, and breast) tissues. Prior to immunostaining, the sections were baked at 50 °C overnight and deparaffinized in xylene and graded ethanol. Antigen retrieval was then performed using citrate buffer pH 6 (ThermoFisher Scientific, Waltham, MA, USA) in decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Sections were stained with anti-SATB1rabbit monoclonal antibody (Clone EPR3895, Epitomics, Burlingame, CA, USA) diluted 1:100 or mouse monoclonal antibody against SATB2 (AMAb90679, CL0320, Atlas Antibodies, Stockholm, Sweden) diluted 1:1,000 in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA) using a commercial kit (UltraVision LP HRP polymer®, Primary Antibody Enhancer, Ultra V Block and DAB plus substrate system®, ThermoFisher Scientific, Waltham, MA, USA). Slides were counterstained with hematoxylin and mounted using Pertex.
Slides were examined, and images were taken using an automated system (VSlide, Metasystems).
4
Immunohistochemical Analysis of Musashi-1 Expression
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Rehydrated samples were thermally treated at 95°C for 20 min in a pre-treatment module (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 1 mM EDTA buffer (pH 8) as an antigen unmasking solution.
A primary polyclonal antibody against Musashi-1 (MSI1) was then applied and incubated at a 1:100 dilution for 16 h at 4°C. Staining with prediluted CD34 (clone QBEnd/10), CD56 (Clone MRQ-42), and TRAP (clone 9C5) for 10 min at room temperature was also performed. Vimentin (clone V9) (1:100 dilution) was used as positive control. A non-immunospecific IgG was used as a negative isotype control. All antibodies were obtained from Master Diagnóstica (Granada, Spain). The immunostaining was developed in an automatic immunostainer (Autostainer480S, Thermo Fisher Scientific, Inc.) using a peroxidase-conjugated micropolymer and diaminobenzidine (Master Polymer, Master Diagnóstica).
Immunopositivity was evaluated semi-quantitatively by a blinded examiner (0=no staining; 1=staining).
Immunopositivity was evaluated in osteocytes, osteoblasts, osteoclasts, mesenchymal stromal cells, and endothelial cells.
A primary polyclonal antibody against Musashi-1 (MSI1) was then applied and incubated at a 1:100 dilution for 16 h at 4°C. Staining with prediluted CD34 (clone QBEnd/10), CD56 (Clone MRQ-42), and TRAP (clone 9C5) for 10 min at room temperature was also performed. Vimentin (clone V9) (1:100 dilution) was used as positive control. A non-immunospecific IgG was used as a negative isotype control. All antibodies were obtained from Master Diagnóstica (Granada, Spain). The immunostaining was developed in an automatic immunostainer (Autostainer480S, Thermo Fisher Scientific, Inc.) using a peroxidase-conjugated micropolymer and diaminobenzidine (Master Polymer, Master Diagnóstica).
Immunopositivity was evaluated semi-quantitatively by a blinded examiner (0=no staining; 1=staining).
Immunopositivity was evaluated in osteocytes, osteoblasts, osteoclasts, mesenchymal stromal cells, and endothelial cells.
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