Dm 200 led light microscope

Skin biopsies were obtained from the untreated healthy paw and untreated inflamed paw at 6 h after intraplantar injection of the 1% carrageenan and the paw of animals challenged under the same hypobaric stress conditions employed in the antiinflammatory assay studies. The paw nonglabrous skin samples were fixed with 4% neutral-buffered formalin for 24 h at 4 °C then were cut in strips, embedded in O.C.T., and frozen as described for the confocal imaging section. Cross-section slices of 10-µm thickness were obtained using a Bright Model OTF cryostat (Bright Instruments). The samples were then stained following the Ellis hematoxylin and eosin staining protocol (57 (link)) and dehydrated with different volumes of ethanol (75%, 95%, and 100%) and xylene before being mounted in DPX and covered with glass coverslips. The samples were analyzed using a Leica DM 200 LED light microscope (Leica Microsystems) equipped with a Leica digital camera (model DFC 295) at a magnification of 20×. Images were processed using Las v4.4 Imaging Software (Leica Microsystems).

Following chondrogenic differentiation, pellets were frozen in OCT embedding matrix (Sakura). Histological staining was performed with haematoxylin and eosin (HE), safranin-O (SO) and toluidine blue (TB). Immunohistochemical labeling was performed for monoclonal antibodies for collagen type-II (col-II) (1:50, Abcam) and aggrecan (1:100, Abcam) in cryosections of 4 µm of each pellet. Secondary antibodies were detected using a polymer-labelled HRP complex (EnVision Detection System Peroxidase/DAB Kit, DAKO). The samples were analyzed using a Leica DM 200 Led light microscope (Leica Microsystems, Madrid, Spain) equipped with a Leica digital camera (Model ICC50W), at magnifications of 4× and 20×. The percentage of immunopositive area was calculated with ImageJ software (version 1.51K, U. S. National Institutes of Health, Bethesda, MD, USA) and normalized against pellet area.

Skin samples, challenged under the same hypobaric stress conditions employed in the permeation studies, were cut into small pieces and fixed with 10% neutral-buffered formalin for 24 h at room temperature. Both porcine and rat skin samples were used in these studies. These samples were subsequently embedded in O.C.T. [26] . Cross-section slices of 20 μm thickness were obtained using a Bright Model OTF cryostat (Bright Instruments, Huntingdon, UK). The samples were stained following the Ellis Haematoxylin and Eosin (H&E) staining protocol [27] and dehydrated with different volumes of ethanol (75%, 95%, and 100%) and xylene for 5 min each, before being mounted in DPX and covered with glass cover slips. The samples were analysed using a Leica DM 200 Led light microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica digital camera (Model DFC 295) at a magnification of 4 × and 40 ×. Images were processed using Las v4.4 Imaging Software (Leica Microsystems, Wetzlar, Germany).