Fixatives

Postnatal day 30 (P30) TWI mice and their WT littermates (5 for each experimental group processed in 5 different experimental sessions, every TWI with its WT littermate) and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution (4% paraformaldehyde and 0.1%–1%–2.5% glutaraldehyde in phosphate buffer, pH 7.4). Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution.
Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm³ in volume. Sciatic nerves were processed without further sectioning.
The selected tissues were further treated for epoxy resin embedding as previously described⁴³ . Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome (UC7, Leica Microsystems, Vienna, Austria) and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.

The most broadly useful contrast stains tested so far are inorganic iodine and phosphotungstic acid (PTA)[22 (link)]. The formulations and general procedures used are given in Table 2, and notes on the fixatives used are in Table 3[23 -25 (link)]. The stains and procedures are simple and the procedures are robust. The staining times were found not to be critical, as long as the stain had sufficient time to penetrate the tissues. Inorganic iodine in alcoholic or aqueous solution diffuses rapidly into fixed tissues and was able to stain most specimens in a few hours or less, although staining was generally done overnight. PTA is a much larger molecule [26 (link)], and the solution used here was found to require overnight incubation to penetrate specimens 2–3 mm thick, and longer for larger specimens. PTA is known to bind heavily to various proteins and connective tissue [27 ,28 ], and this property, along with electron-shell energies that match common x-ray source emissions, suggested that it might be a useful stain for x-ray imaging. A few samples were tested with phosphomolybdic acid (PMA) staining, used similarly to PTA. The results (not shown) were generally similar, and PMA was not pursued further here (but see refs. [29 (link)] and [30 ] for successful application of PMA).

Sixty-one patients with active IBD (24 UC, 19 CDc and 18 CDi), refractory to corticosteroids and/or immunosuppression, and a control group of 12 individuals (6 colon and 6 ileum) who underwent endoscopy for screening for polyps were studied. The patients underwent endoscopy with biopsies from diseased bowel (colon for UC and CDc, and ileum for CDi) within a week prior to the first intravenous infusion of 5 mg infliximab per kg body weight. They underwent a second endoscopy with biopsies 4 weeks after the first infliximab infusion in case of a single infusion and at 6 weeks if they received a loading dose of infliximab at weeks 0, 2 and 6. The biopsies were taken at sites of active inflammation but at a distance of ulcerations. In the case of healing at control endoscopy, the biopsies were obtained in the areas where lesions were present before therapy. The endoscopist was not blinded to treatment. Half of the biopsies were immediately snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation and/or immunohistochemistry, except for the biopsies from 1 CDc patient after infliximab treatment which were of poor technical quality. The residual biopsies were fixed in Carnoy's fixative for up to 5 hours and then dehydrated, cleared and paraffin-embedded for histologic examination. The features of chronic intestinal inflammation were scored in haematoxylin-eosin stained slides from the paraffin blocks of each patient using a previously reported scoring system for UC [26] (link) and for CD [8] (link). The pathologists who scored the biopsies (KG and GDH) were blinded to treatment.
The response to infliximab was assessed 4 to 6 weeks after the first infliximab treatment. For UC and CDc, the response to infliximab was defined as a complete mucosal healing with a decrease of at least 3 points on the histological score for CDc [8] (link) and as a decrease to a Mayo endoscopic subscore of 0 or 1 with a decrease to grade 0 or 1 on the histological score for UC [26] (link), [27] (link). Patients who did not achieve this healing were considered non-responders although some of them presented endoscopic and/or histologic improvement. Of the 43 colonic IBD (IBDc) patients, we identified 20 responders (8 UC and 12 CDc) and 23 non-responders (16 UC and 7 CDc). If the same response criteria of CDc were used for CDi, only one patient showed complete endoscopic and histologic healing. Therefore, we had to use less strict response criteria for CDi. Patients with a clear improvement of the ulcerations and a decrease on the histological score [8] (link) were considered responders. Of the 18 CDi patients, we identified 8 (partial) responders and 10 non-responders.
The baseline characteristics of the patients are summarized in table 1.

Colonic tissues were fixed with 2.5% glutaraldehyde. After removal of excess fixative with PBS, samples were fixed with 1% osmic acid at 20 °C for 2 h, dehydrated in acetone, and then infiltrated with acetone and epoxy. Epoxy resin-embedded tissues were sectioned (80 nm in thickness), stained with uranyl acetate and lead citrate, and finally viewed under a transmission electron microscope (FEI, Hillsboro, America).

The toxicity of repeated and high dose of CNPs treatment was assessed by histological and hematological analyses. Briefly, Cy5.5-CNPs (10, 22.5 or 90 mg/kg) were intravenously injected into BALB/c mice with single- or multi-dosage (three times). On day 7 after treatments, major organs (liver, lung, spleen, kidney, brain and heart) were collected from mice, and structural abnormalities in organ tissues were assessed by staining with H&E. In the case of hematological analyses, blood samples were collected from the mice on day 7 and centrifuged at 2200 rpm to obtain plasma. The following factors in blood samples were measured; alanine aminotransferase (ALT), blood urea nitrogen (BUN), alkaline phosphatase (ALP), aspartate Aminotransferase (AST), creatine kinase (CK) and troponin I. The cardiotoxicity by Cy5.5-CNPs was further analyzed after multiple-dosage. The heart tissues were collected from mice after treatment with 10, 22.5 or 90 mg/kg of Cy5.5-CNPs three times. The accumulation of Cy5.5-CNPs in heart tissues was observed using a Leica TCS SP8 confocal laser-scanning microscope. Collagen fiber in heart tissues were stained with Masson's trichrome. Briefly, heart tissues were incubated in Bouin's fixative for 30 min at 56 °C, and the nuclei were co-stained with Weigert's iron hematoxylin. Then, cytoplasm was stained with Biebrich scarlet-acid fuchsin, and then differentiated in phosphomolybdic–phosphotungstic acid. The collagen matrix in heart tissues was stained with aniline blue solution. The collagen in heart tissues were quantitatively analyzed using an Image Pro software, and collagen contents were presented in proportion to the total area of heart tissues.

All specimens used for sequencing and experimentation were collected from Yunnan Province, China. All vouchers are stored in the Herpetological Museum of the Chengdu Institute of Biology, Chinese Academy of Sciences.
For the TEM experiments, two fresh skin samples (1cm×1cm) per color morph from the Asian vine snake were collected. The samples were then cut into small pieces (1 mm³) in fixative. The tissue blocks were transferred to an Eppendorf tube with fresh TEM fixative for further fixation, then washed using 0.1 M PB (pH 7.4) three times (15 min each). The samples were dehydrated in an increasing ethanol series at room temperature, followed by two changes of acetone and transfer to resin for embedding. The resin blocks were cut to 60–80-nm slices on an ultra-microtome (Leica, UC7), and the ultra-thin sections were put onto the 150-mesh cuprum grids. The cuprum grids were then stained with 2% uranium acetate-saturated alcohol solution and 2.6% lead citrate, respectively. Finally, the cuprum grids were observed under a TEM (Hitachi, HT7800/HT7700) and imaged.