Axioskop 2 light microscope

Immunohistochemistry was performed as described (37 (link)). Human sections (30–40 µm) were pre-incubated for 10 min with 5% H2O2 in 5% methanol in PBS, and for 24–48 h in PBS-0.3% Triton X-100, 2% normal donkey serum (NDS) at 4°C, with goat anti-P2X7 receptor antibody (1:100, peptide YETNKVTRIQSMNY-C from the N-terminus of human P2RX7 corresponding to amino acid residues 13-26, MyBioSource, San Diego, CA, USA). Sections were then incubated with biotinylated donkey anti-goat antibodies (Jackson ImmunoResearch Europe Ltd., Suffolk, UK), followed by avidin–biotin–peroxidase reactions (Vectastain, ABC kit, Vector, Burlingame, CA, USA), using 3,3′-diaminobenzidine (Sigma-Aldrich) as a chromogen. Some sections were counterstained with Luxol fast blue, in order to detect lesion types. Sections were mounted on poly-lysine slides and air dried for 24 h. The histological preparations were examined using an Axioskop 2 light microscope (Zeiss). Images were taken with Neurolucida software (MBF Bioscience, USA).

In total, 72 samples were palynologically analysed. Pollen extraction of 1 mL sediment was performed following the standard procedure of [72 ] including HCL, KOH, HF (including boiling) and actetolysis. A Lycopodium spore tablet (Batch no. 1031; n = 20,848 ± 1460) was added to each sample to estimate the pollen concentration [73 ]. Pollen slides were analysed with a Zeiss Axioskop 2 light microscope (400–600× magnification). At least 300 pollen grains were identified in each sample. Taxonomic identification was based on published pollen and non-pollen palynomorph atlases [74 ,75 ,76 ,77 ,78 ,79 ,80 ,81 (link),82 (link),83 (link),84 ] and a pollen reference collection at the Arctic and Antarctic Research Institute (Saint Petersburg) and the Alfred Wegener Institute (Potsdam). The systematic placement of Chenopodiaceae changed after the application of molecular techniques and is now included into Amaranthaceae [85 ]. To facilitate comparability with previous palynological studies in the Laptev region, we use Chenopodiaceae in this manuscript.

Slides were examined using a Zeiss Axioskop 2 light microscope. Bregma levels were determined for each slice, using a rat brain atlas for each brain slice to allow the identification of subregions of the hypothalamic area. Images were taken using a QImaging Fast 1394 Camera. Images were collected using a GFP filter to visualize fluorescent neurons, and DAB reactivity was visualized using bright-field microscopy.

All patients had open deltoid or quadriceps muscle biopsy. Muscle specimens were conventionally processed [3 (link),27 (link)]. One muscle sample was flash-frozen in isopentane cooled by liquid nitrogen for histoenzymology and immunohistochemistry, another fixed in formaldehyde fixative for routine histology, and the last processed for electron microscopy. Unfixed 7 μm cryosections were routinely stained with hematein-eosin (HE), Gomori or Masson trichrome, PAS, Sudan black, and histoenzymological reactions for NADH-TR and cytochrome-C-oxidase (Cox).Myosinolysis, microinfarctus and perifascicular atrophy were assessed as previously proposed [28 (link),29 ]. Immunoperoxidase investigations were carried out on frozen 7 μm sections using a Bond-III automaton (Leica, Nanterre, France) to detectHLA-ABC, HLA-DR, membrane attack complex (MAC/C5b-9), regenerating myofibers (CD56/neural cell adhesion molecules [NCAM]), macrophages (CD68), and other leukocyte subsets CD3, CD4, CD8 CD20. Detailed characteristics of primary antibodies are in Additional file 1: Table S1. Morphological analyses were carried out on Axioskop® 2 light microscope (Zeiss) and digital image capture using Coolscope® (Nikon) device.

In-vitro wound healing scratch assay was performed using confluent monolayer corneal epithelial cells. Primary human corneal epithelial cells were grown to confluence on collagen coated plates. For migrating group of cells, a linear scratch a cell free area (abrasion 1 mm) was made using a sterile 200-lL pipette tip^{33 (link)} while unscratched cells (non-migrating) were used as control group. Immediately after the scratch at 0, 6, 12, 18, 24, 36, 42 and 48 hours images of the scratch area were captured using the Zeiss AxioCam MR digital camera on a Zeiss Axioskop2 light microscope (Carl Zeiss). The remaining wound area was measured using ImageJ software, provided in the public domain at http://rsb,info.nih.gov/ijnih.gov, Bethesda, MD.

Macrophages (3 × 10⁵) from wild-type mice were seeded in 48-well plates. After overnight incubation, cells were pretreated with 0, 1 or 7.5 μM of cytochalasin D for 30 min and then incubated with unbaked calculus for 8 h. The cells were immobilized in iPGell and fixed in 0.05 M cacodylate buffer (pH7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde, post-fixed with osmium tetraoxide in 0.05 M cacodylate buffer (pH7.4), and embedded in Epon-Araldite resin. For analysis of the uptake of dental calculus in the macrophages, semithin sections (1 μm) were stained with toluidine blue and examined with a Zeiss Axioskop 2 light microscope (Zeiss, Oberkochen, Germany) at 1000 times magnification. Ultrathin sections (0.1 μm) were stained with 2% uranyl acetate and Reynold’s lead citrate, and examined under a Hitachi H-7100 electron microscope (Hitachi High-Technologies, Tokyo, Japan).