Envision peroxidase system

Formalin-fixed, paraffin-embedded, de-waxed sections 4 µm thick of the frontal cortex (GM) and subcortical WM in five MA cases were processed for specific immune-histochemistry. The sections were boiled in citrate buffer (20 min) to retrieve protein antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂ O₂ solution (15 min) followed by 3% normal horse serum solution. Then the sections were incubated at 4ºC overnight with one of the primary rabbit polyclonal antibodies: 3-ketoacyl-CoA thiolase (ACCA1) (MyBioSource MBS1492126) used at a dilution of 1/100; Fatty Acid Synthase (FAS) (C20G5, Cell Signaling 3180) diluted 1/50, and Stearoyl-CoA desaturase (SCD) (MyBioSource, BS421254) used at a dilution of 1/50. After incubation with the primary antibody, the sections were incubated with EnVision+ system peroxidase (Dako, Agilent Technologies, Santa Clara, CA, USA) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂ O₂. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Sections were slightly counterstained with hematoxylin. Due to the individual variability of the immunostaining, no attempt at quantification was performed; immunohistochemistry was used to assess the localization of the enzymes.

Paraffin sections were processed for immunohistochemistry using the AT8 (Innogenetics, Ghent, BE) and Aβ (Dako, Glostrup, DK) antibodies, both at a dilution of 1:50, the binding of which was detected with EnVision+ System peroxidase (Dako, Agilent Technologies, Barcelona, Spain). The immunoreaction was visualized with diaminobenzidine and H₂O₂, and the sections were counterstained lightly with haematoxylin (bar = 50 μm). The patients’ AD pathology was categorized using the Braak and Braak scale adapted to immunohistochemistry on paraffin sections.

De-waxed sections, 4μm thick, of the lumbar spinal cord (n=6 sALS, n=6 controls) and frontal cortex area 8 (n=6 sALS, n=6 controls) were processed in parallel for immunohistochemistry. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂O₂ for 15min followed by 3% normal horse serum. Then the sections were incubated at 4°C overnight with anti-YKL40 primary antibody (PA5-43746, ThermoFisher, Waltham, Massachusetts, USA) at a dilution of 1:200. Immediately afterwards, the sections were incubated with EnVision + system peroxidase (Dako, Agilent, Santa Clara, CA, USA) for 30min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂O₂. No signal was obtained following incubation with only the secondary antibody. Sections were slightly stained with haematoxylin.

The hippocampus was selected for study in human cases. The whole hemisphere was assessed in mice. De-waxed sections, 4 µm thick, were processed for immunohistochemistry. The sections were boiled in citrate buffer at pH 6 (20 min) to retrieve tau antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol–1% H₂O₂ solution (15 min), followed by 3% normal horse serum solution. The sections were incubated at 4 °C overnight with one of the primary antibodies listed in Table 1. After incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase (Dako-Agilent, Barcelona, Spain) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine (DAB) and H₂O₂. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Deposits in neurons and related axonal fibers and threads in the hippocampus in inoculated mice are expressed semi-quantitively using the morphometric approach.

For immunohistochemistry, slides containing sections of mice and human intestinal tissues (4 µm) were deparaffinised, rehydrated and processed as previously described^{31 (link)}. Incubation with primary antibody was carried out in a wet chamber overnight at 4 °C. Hakai dilution was 1:700 and FASN dilution was 1:500. Commercial kit for immunohistochemistry was Dako EnVision + System, Peroxidase (EnVision + System, HRP) purchased to Agilent Technologies, Inc. Nuclei were counterstained with Gill´s haematoxylin and mounted with DePeX. Pictures were taken with an Olympus BX50 microscope. Quantification of HRP signals was performed with ImageJ software by analysing 5 photographs of each sample, and the represented results are shown as mean ± SEM.

De-waxed sections, 4 microns thick, were processed for immunohistochemistry. The sections were boiled in citrate buffer pH = 6 (20 min) to retrieve tau antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂O₂ solution (15 min) followed by 3% normal horse serum solution. The sections were incubated at 4 °C overnight with one of the primary antibodies listed in Table 2. Following incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase (DakoAgilent, Barcelona, Spain, DK) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂O₂. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Lesions were assessed in consecutive sections within coordinates −2.3 to −1.6 interaural, and −1.4 to −2.1 from Bregma.