Hm525

Anesthetized rats (4 /group) were perfused with 300 ml 0.9% NaCl and 4% paraformaldehyde in phosphate-buffered saline (PBS), and the extracted brains were stored with 4% paraformaldehyde and then 30% sucrose for 48 hours before being frozen in an embedding compound. The frozen whole brains were cut to a thickness of 30 μm with a cryostat (HM525, Thermo Scientific). The section slides were incubated overnight at 4 °C with primary antibodies against GFAP (1:1000; catalog ab7260, Abcam), NeuN (1:1000; catalog MAB377, Millipore), MAP2 (1:1000; catalog ab11267, Abcam), PSD95 (1:500; catalog ab18258, Abcam), synapsin (1:500; catalog ab64581; Abcam), and CD68 (1:1000; cat MAB1435; Millipore), washed with PBS, and incubated for 2 hours at room temperature with Alexa Fluor 488 and Cy^tm 3-conjugated AffiniPure F(ab’)₂ Fragment Donkey Anti-Mouse IgG secondary antibodies (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). DAPI was used for counterstaining. Immunofluorescent sections were imaged by LSM700 confocal microscope (Zeiss, Oberkochen, Germany) using 10× and 40× PlanApo oil-immersion lens. Briefly, 12 μm-thick confocal Z-stacks of the synaptic zone in ZI were captured. Three image stacks per rat (4 /group) were used for analyses. The number of cells with colocalized GFAP and mGluR1 was quantified. Three images per rat (4 /group) were used for analyses.

Samples of the ganglia of each specimen (cerebro-pleural, pedal, and visceral) were collected using a scalpel, incising the trailing muscles to open the shells of the animals. Immediately after extraction, the samples were placed in a 4% paraformaldehyde (PFA) solution, in a ratio of 0.5 cm³ of tissue to 5 mL of solution for 2–3 h at room temperature (20–22 °C). Then, the samples were washed with phosphate-buffered saline (PBS; 3 × 20 min). The material of each ganglion was transferred to a 30% sucrose solution and left for incubation overnight at 4 °C. The samples were embedded in a freezing medium (OCT cryomount, HistoLab, Finland) and frozen for storage at -20 °C. Tissue cryosections were then obtained using a cryostat HM525 (Thermo Fisher Scientific) with a thickness of 14 μm.

On POD 14, the rats were deeply anesthetized with urethane (1.25 g/kg, i.p.) immediately after repetitive trials of ICS or sham stimulation. The anesthetized rats were transcardially perfused with normal saline (0.9% NaCl) followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4). For postfixation, the brains were soaked in 30% saccharose in phosphate‐buffered saline (PBS) and cryosectioned to a thickness of 20 μm using a cryostat (HM525, Thermo Scientific, Waltham, MA, USA). Brain sections were washed with PBS and blocked in 10% normal donkey serum (017‐000‐121; Jackson ImmunoResearch, West Grove, PA, USA) in 0.3% Triton X‐100 for 1 h at RT. Brain sections were incubated overnight at 4°C with primary antibodies (Table 1) and rinsed with PBS containing 0.3% Tween‐20. A mixture of secondary antibodies (Table 1) was incubated for 2 h at RT. All sections were mounted with 4′,6‐diamidino‐2‐phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). Images were captured using a laser scanning confocal microscope (LSM 700; Carl Zeiss, Jena, Germany). Images were subjected to maximum intensity projection (MIP) using Zen Black software (Carl Zeiss) to represent the branches of glial cells.

For IHC, the rats were perfused with 200 ml 0.9% NaCl and 4% paraformaldehyde in sodium phosphate buffer, and the brains were excised and incubated with 4% paraformaldehyde for 24 hours before freezing in an embedding compound on dry ice. The frozen brains were cut with a cryostat (HM525, Thermo Scientific, Waltham, MA, USA) at a thickness of 30 μm. The sections were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-GFAP (1:500; catalog ab7260; Abcam); and mouse anti-NeuN (1:100; catalog MAB377; Millipore) and then washed with PBS and further incubated for 2 hours at room temperature with Alexa Fluor 488– and Cy^tm 3-conjugated AffiniPure F(ab’)₂ Fragment Donkey Anti-Mouse IgG secondary antibodies (all from Jackson ImmunoResearch, 1:200). DAPI was used for counterstaining. The immunofluorescence images were obtained using a confocal microscope with AxioScan Z1 (Zeiss, Goettingen, Germany) and LSM700 (Zeiss, Goettingen, Germany). The number of cells with colocalized expression of NeuN and GFAP were quantified. Briefly, 12 μm-thick confocal Z-stacks of the synaptic zone in the ACC and M1 cortex were imaged. The maximum projections of 3 consecutive optical sections were generated from the original Z-stack. Three image stacks per rat (n = 4 rats/group) were used for analyses.

To detect the occurrence of transcripts of Fhabl1, in situ hybridization was performed as described earlier with slight modifications (31 (link)). Twelve-week-old F. hepatica worms were embedded in Tissue-Tek (Sakura Finetek), frozen on dry ice, and stored at −80°C until use. Traversal cryosections of 10 μm thickness were prepared using a cryostat HM525 (Thermo Fisher Scientific, Germany). Sections were dried, post-fixed in 4% PFA, and permeabilized with PBSTx. The hybridization reaction was carried out overnight at 55°C. Following washing with saline sodium citrate buffer (SSC), the sections were incubated with anti-DIG antibodies coupled with alkaline phosphatase (Roche, Germany). After subsequent washing steps with maleic acid buffer with Tween (MAB-T), the development reaction was carried out using naphthol-AS-phosphate, and Fast Red TR (Sigma). The Riboprobes for the hybridization reaction were generated as previously described (32 (link)). The following primers were used to generate the template for probe synthesis with a length of 596 bp: 5′-GAATCTCCTTCTCCTAACGGT-3′, reverse 5′-ACCAGATTTTTTAGGAGGTCTTC-3′. The labeling reaction using digoxigenin-11-UTP (NU-803-DIGXS; Jena Bioscience, Germany) was done using T3 or SP6 RNA polymerases for sense and antisense probes. Labeled transcripts were controlled for the correct size by gel electrophoresis.

For immunhistochemistry mice were deeply anesthetized by a intraperitoneal injection of a mixture of ketamine (280 mg/kg bodyweight) and xylacine (20 mg/kg body weight). Access to the heart was obtained by removing part of the rips together with the sternum and lung displacement. Subsequently an injection needle connected to a perfusion pump punctured the left ventricle, and the right atrium was opened with small scissors. Perfusion was started immediately by a perfusion pump. Approximately 200 mL of fixative (Ringer solution + 4% paraformaldehyde) was pumped through the vascular network of the mouse.
After perfusion the transplanted eye was isolated and postfixed in the same fixative for another 4 hr. After washing (3x, 2 hr each) the eye was incubated in 30% sucrose solution over night (4°C) before embedding in Tissue-Tek O.C.T. compound (Sakura Finetek Europe B.V.). For embedding the eye was placed in a small beaker and surrounded by Tissue-Tek O.C.T. compound, dipped for 1 min into 2-methylbutane at −80°C and stored at −80°C until cutting. A cryostat (Thermo Scientific HM525) was used to produce 10–20 µm thick eye slices. Slices were collected on Superfrost+ slides (VWR). Staining with primary and secondary antibodies followed standard protocols. Samples were mounted using ProLong Gold Antifade (RRID:SCR_015961) as a mounting medium.