For IL1β and CD68 immunohistochemistry, cryostat sections (7 μm) were fixed with 4% PFA/PBS(10 min, ambient temperature). Non-specific sites were blocked with 1% normal swine serum (Dako Deutschland GmbH, Hamburg, Germany) in PBS. Single staining was performed by incubation of polyclonal anti- IL1ß (1:100: Abcam; Cambridge, UK) or monoclonal anti-CD68 primary antibody (1:50; AbD Serotec, Kidlington, UK) with goat polyclonal HRP-conjugated anti-rabbit (1:200; LINARIS GmbH, Dossenheim, Germany) or anti-rat (1:100; AbD Serotec, Kidlington, UK) antibody; endogenous peroxidase activity was suppressed with 3 % H2O2 in PBS; afterwards, the sections were incubated with DAB solution (Roche Diagnostics). Nuclei were counterstained with Mayer’s hematoxylin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).
Dab solution
Manufactured by Roche
Sourced in United Kingdom, Switzerland, Germany
The DAB solution is a laboratory reagent used in immunohistochemistry and in situ hybridization techniques. It serves as a chromogenic substrate that produces a brown colored precipitate upon reaction with the enzyme-labeled detection system. The DAB solution enables the visualization and localization of target molecules within biological samples.
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4 protocols using dab solution
1
Immunohistochemical Analysis of IL1β and CD68
2
Histological Analysis of APAP-Induced Liver Injury
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After isolating the livers from the APAP-induced acute liver injury models, the livers were fixed with 10% neutral buffered formalin. The liver tissues were embedded with paraffin, and 5 μm sections were cut from them. They were stained with hematoxylin and eosin for histopathological analysis. Fixed liver sections were also used for TUNEL assay after they were incubated with terminal deoxynucleotidyl transferase dUTP nick and labeled with enzyme for 1 h at 37 °C in the dark. After the slides were washed three times in PBS for 3 min, they were incubated with POD substrate for 30 min at 37 °C in the dark. TUNEL-positive cells were detected using DAB solution (Roche, Basel, Switzerland). Necrotic cells were evaluated using a Leica ICC50 HD microscope (Leica Microsystems, Wetzlar, Germany) with magnification of × 200.
3
Quantifying Muscle Capillary Density
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Cryo cross-sections of gastrocnemius or soleus muscle were cut in a cryostat, fixed in 4% PFA/PBS 10 min, endogenous peroxidase was blocked with 3% H2O2, and washed in phosphate-buffered saline (PBS; pH 7.4) at room temperature. Sections were then incubated 30 min at 37°C with 40 μg/ml in PBS HRP-conjugated Isolectin B4 of Bandeiraea simplicifolia (BSI- B4) (Sigma-Aldrich Co. LLC, ST. Louis, USA). Afterwards sections were washed with PBS and incubated with DAB solution (Roche Diagnostics), rinsed with PBS, counterstained with Mayer's hematoxylin (Carl Roth GmbH, Karlsruhe) and cover-slipped in mounting medium for histology, DePeX (SERVA Electrophoresis GmbH, Heidelberg, Germany). Nuclei were counterstained with Mayer's hematoxylin (Carl Roth GmbH). Capillary with a diameter of ≤ 5μm were identified in cross-sections by brown staining and quantified using the software software ImageJ (Scion Image, National Institutes of Health, Bethesda, USA). The average capillary numerical density (number of capillaries/mm2) and capillary contact per muscle fiber (number of capillaries/fiber) was calculated for each muscle cross-section. To analyze neuromuscular junctions α-Bungarotoxin (α-BT) histochemistry was performed (Materials and methods A in S1 File ).
4
Detection of Recombinant TcpA Protein
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For Western blot analyses, 0.5 μg of purified recombinant TcpA protein was used per well. As a negative control, the bacterial lysate from induced E. coli BL21 (DE3) contain pGEX4T1 vector was analyzed by Western blot.
The gel was blotted on to Poly vinylidine difluoride (PVDF Membrane, Roche Diagnostic) membrane using transfer buffer containing 25 mM Tris (pH 8.3), 192 mM glycine and %20 methanol at 90 volts for 1.5 hours at 4°C. The blotted membrane was blocked with 2.5% (w/v) BSA in TBST buffer (0.5M NaCl, 0.02 M Tris pH 8.5, 0.05% Tween 20) for 1 hour at room temperature. Membranes were incubated for 2 hours at room temperature with rabbit, mice sera and patient serum, diluted 1:100 and 1:50, respectively. Normal sera from rabbit, mice and human were used as controls. After reactions with the primary antibody, the blots were washed three times with TBST and incubated with peroxidase conjugated goat anti-rabbit IgG , anti mouse IgG and antihuman Ig (G, A, M) at a 1:2500 dilution in TBST. The blots were then washed three times with TBST and reactions were developed by diamino Banzidine (DAB) Solution (Roche Diagnostic).
The gel was blotted on to Poly vinylidine difluoride (PVDF Membrane, Roche Diagnostic) membrane using transfer buffer containing 25 mM Tris (pH 8.3), 192 mM glycine and %20 methanol at 90 volts for 1.5 hours at 4°C. The blotted membrane was blocked with 2.5% (w/v) BSA in TBST buffer (0.5M NaCl, 0.02 M Tris pH 8.5, 0.05% Tween 20) for 1 hour at room temperature. Membranes were incubated for 2 hours at room temperature with rabbit, mice sera and patient serum, diluted 1:100 and 1:50, respectively. Normal sera from rabbit, mice and human were used as controls. After reactions with the primary antibody, the blots were washed three times with TBST and incubated with peroxidase conjugated goat anti-rabbit IgG , anti mouse IgG and antihuman Ig (G, A, M) at a 1:2500 dilution in TBST. The blots were then washed three times with TBST and reactions were developed by diamino Banzidine (DAB) Solution (Roche Diagnostic).
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