Cy3 conjugated donkey anti rabbit igg

After cryosection, 2 μm thick brain sections were mounted onto glass slides. Sections were washed with PBS (pH 7.4), followed by antigen retrieval using sodium citrate buffer, pH 6, at 95°C for 25 min in a water bath. Nonspecific staining in sections was blocked using 10% donkey serum in 0.1% Tween 20-PBS for 1 h at room temperature. Primary antibodies were added overnight at a dilution of 1 : 350 for goat Iba-1 (Abcam), 1 : 1,000 for rabbit MMP-2 (Abcam) at 4°C. Alexa 488-conjugated donkey anti-goat IgG (1 : 200, Jackson Lab, Suffolk, United Kingdom) or Cy3-conjugated donkey anti-rabbit IgG (1 : 200, Jackson Lab) was subsequently applied. The nuclei were counterstained with DAPI (Sigma-Aldrich). Images were taken using a microscope (Axio Imager Z1, Carl Zeiss, China). This part was similar with the methods of Hu et al. [32 (link)].

The determination of intra-epidermal nerve fiber density (IENFD) from skin biopsy samples is a validated and sensitive diagnostic tool for the assessment of small fiber pathology. Biopsy samples were taken in accordance with the consensus document produced by the European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the utilization of skin biopsy samples in the diagnosis of peripheral neuropathies (Lauria et al., 2010 (link)). Skin biopsies were taken with a disposable 3mm punch biopsy circular blade (Stiefel Laboratories Inc, GSK Plc) from 10 cm proximal to the lateral malleolus and from the lateral aspect of the proximal thigh. Nerve fibers were visualized using rabbit anti-PGP9.5 antibody (Zytomed, 1:200) with Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152, 1:1000). The nerve fibers are counted as they cross the epidermal-dermal junction in order to quantify intra-epidermal nerve fiber density. Images were taken using an LSM 700 microscope with a Plan-Apochromat objective (Carl Zeiss) at 40 × and 63 × magnification.

Rats were deeply anesthetized with 0.4% sodium pentobarbital anesthesia (40 mg/kg
body weight, i.p.) and perfused transcardially with 500 ml of cold
phosphate-buffered saline (PBS) followed by 500 ml of cold 4% paraformaldehyde
(PFA). The spinal cord was removed and postfixed with the same 4% PFA for 1–2 h
at 4°C and then transferred to 30% sucrose in PBS overnight. Sample sections (20
μm thickness) were adhered on gelatin-coated glass slide with a cryostat
(Leica). The sections were washed three times with PBS pH = 7.35–7.40 and then
hatched in PBST (0.3% Triton in PBS) for 40 min, blocked with QuickBlock buffer
(Beyotime, P0260) for 10 min, subsequently incubated overnight at 4°C with
primary antibody for rabbit-anti-p-PKC (1:200, Abcam),
mouse-anti-CGRP (1:200, Abcam), goat-anti-GFAP (1:200, Abcam),
mouse-anti-OX-42(1:200, Abcam), anti-IB4(1:50, Sigma), and mouse-anti-NeuN
(1:200, Millipore). The sections were then incubated for 60 min at room
temperature with secondary antibodies Cy3-conjugated donkey anti-rabbit IgG
(1:200, Jackson ImmunoResearch), FITC-conjugated donkey anti-goat IgG (1:200,
Jackson ImmunoResearch), and Alexa 488-conjugated donkey anti-mouse IgG (1:200,
Thermofisher). The stained sections were captured with LSM 780 (Carl Zeiss). The
fluorescent density was quantified with a computer-assisted imaging analysis
system (ImageJ, National Institutes of Health).