Mice were euthanized under deep anesthesia (ketamine 180 mg/kg; xylazine 16 mg/kg) by intracardiac perfusion with phosphate-buffered saline (PBS) followed by perfusion with 4% (w/v) paraformaldehyde (PFA) solved in PBS. Brain and spinal cord were removed and fixed in 4% PFA overnight. Prior to paraffin embedding, the spinal cord was cut into seven to ten transverse segments (3 mm thick) and coronal brain cuts were made. Sections (3 μm) were stained by HE (hematoxylin and eosin) and LFB-PAS (luxol fast blue including periodic acid-Schiff). Immunohistochemistry was performed using a biotin-streptavidin peroxidase technique (Dako) and an automated immunostainer (AutostainerLink 48, Dako). Sections were pretreated with citrate buffer (pH 6) in a steamer for immunohistochemistry for Mac3 (clone M3/84, #550292, 1:200 also known as CD107b or LAMP-2; BD Pharmingen). Quantification of demyelination and Mac3 infiltration was performed as described previously67 (link),68 (link).
Biotin streptavidin peroxidase technique
Manufactured by Agilent Technologies
Sourced in Italy
The Biotin-streptavidin peroxidase technique is a laboratory method used for the detection and visualization of target molecules in biological samples. It involves the use of biotin-labeled probes that bind to the target, and streptavidin-conjugated peroxidase enzyme that catalyzes a colorimetric reaction, allowing the visualization of the target.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer link 48, by Agilent Technologies (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Clone m3 84, by BD (1 mentions)
Lamp2, by BD (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Axio observer, by Zeiss (1 mentions)
Bz 2 analyzer, by Keyence (1 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
Anti tgf β1, by Merck Group (1 mentions)
Anti il 13, by Santa Cruz Biotechnology (1 mentions)
Anti il 4, by R&D Systems (1 mentions)
4 protocols using biotin streptavidin peroxidase technique
1
Histological Analysis of Demyelination and Inflammation
2
Histological Analysis of Mouse Tissues
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Mice were sacrificed under deep anaesthesia by intracardiac perfusion with PBS followed by perfusion with 4% (w/v) paraformaldehyde (PFA) dissolved in PBS. Organs were removed and fixed in 4% PFA overnight; except the small intestine, which was flushed with PBS, opened longitudinally, and rolled up starting with the distal part before fixation (Swiss-rolling technique). Prior to embedding in paraffin, the spinal cord was cut into 7–10 transverse segments (3-mm thick) and coronal brain cuts were made. Sections (3 µm) were stained by haematoxylin and eosin and Luxol fast blue including periodic acid-Schiff (LFB-PAS). Immunohistochemistry was performed using a biotin-streptavidin peroxidase technique (Dako) and an automated immunostainer (AutostainerLink 48, Dako). Sections were pretreated with citrate buffer (pH 6 or 9) in a steamer. The primary antibodies were specific to Mac3 (clone M3/84, also known as CD107b or LAMP-2; BD Pharmingen) or CD3 (MCA, Serotec). 3,3′-diaminobenzidine (Dako) was used as a colour substrate and sections were mounted with Eukitt® mounting medium (O. Kindler GmbH) after dehydration. Images were acquired with the microscope AxioObserver (Carl Zeiss) or BioRevo BZ-9000 (Keyence) using the software AxioVision or BZ-II Analyzer (Keyence).
3
Quantifying Extracellular Matrix Proteins
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For indirect immuno-fluorescence (IIF), 1.5 × 104 cells were incubated with anti-collagen type I (COL1A1), anti-cellular fibronectin, and anti-α-SMA followed by goat anti-mouse FITC-conjugated antibody. Nuclei were visualized using Hoechst 33342 (all from Sigma-Aldrich, Milano, Italy). For immuno-cytochemistry (ICC), 1.5 × 104 cells were incubated with anti-TGF-β1 (Sigma-Aldrich) and then immune-stained using the streptavidin–biotin–peroxidase technique (Dako Cytomation, Milano, Italy) and incubated with 3,3-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin. To quantify the expression of the proteins, the percentage of area occupied by the protein inside the cells was calculated using Fiji-ImageJ software (available online at https://imagej.net/Fiji ) [22 (link)].
4
Immunostaining Analysis of IL4 and IL13
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Considering the action of Dupilumab on IL4 and IL13, immunocytochemical and immunofluorescence analyzes for IL4 and IL13 were performed on C-MSC and AD-MSC at T0 and T16.
For Immuno-cytochemistry (ICC), 1.5 × 104 cells were incubated overnight with anti-IL4 (R & D Systems, Minneapolis, Canada) or anti-IL13 (Santa Cruz Biotechnology, Dallas, TX, USA) primary antibody. Then, cells were immune-stained using the streptavidin–biotin–peroxidase technique (Dako Cytomation, Milano, Italy) and incubated with 3,3-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin.
For Indirect Immuno-Fluorescence (IIF), we incubated the same number of cells with the anti-IL4 or anti-IL13 antibody, followed by goat anti-mouse FITC-conjugated antibody. Nuclei were visualized using Hoechst 33342 (all from Sigma-Aldrich, St. Louis, MO, USA).
To quantify the expression of the proteins, the percentage of area occupied by the protein within the cells was calculated using Fiji-ImageJ software [22 (link)].
For Immuno-cytochemistry (ICC), 1.5 × 104 cells were incubated overnight with anti-IL4 (R & D Systems, Minneapolis, Canada) or anti-IL13 (Santa Cruz Biotechnology, Dallas, TX, USA) primary antibody. Then, cells were immune-stained using the streptavidin–biotin–peroxidase technique (Dako Cytomation, Milano, Italy) and incubated with 3,3-diaminobenzidine. Slides were counterstained with Mayer’s hematoxylin.
For Indirect Immuno-Fluorescence (IIF), we incubated the same number of cells with the anti-IL4 or anti-IL13 antibody, followed by goat anti-mouse FITC-conjugated antibody. Nuclei were visualized using Hoechst 33342 (all from Sigma-Aldrich, St. Louis, MO, USA).
To quantify the expression of the proteins, the percentage of area occupied by the protein within the cells was calculated using Fiji-ImageJ software [22 (link)].
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