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Immunohistochemistry of the Pulmonary Extracellular Matrix
Published in Joan Gil, Models of Lung Disease, 2020
Antonio Martinez-Hernandez, Peter S. Amenta
Freezing results in enough tissue hardening to permit cryostat sectioning at 5-10 μm, suitable for light microscopy or, after subsequent embedding in plastic, for electron microscopy (Martinez-Hernandez, 1987a,b; Amenta and Martinez-Hernandez, 1987). With a cryoultramicrotome, thin sections can be obtained directly, obviating the use of organic solvents (Tokuyasu, 1984). Whatever the final use of frozen blocks, it is essential to minimize ice crystal formation. To this end, small blocks, the addition of cryoprotectors, and a fast freezing rate are critical (Martinez-Hernandez, 1987a,b; Amenta and Martinez-Hernandez, 1987). Frozen sections, while not providing the optimal preservation, have some advantages; for instance, the antigenic determinants are directly available. In contrast, with plastic or paraffin, the embedding media remains in the sections (as a barrier to antibody penetration) or must be removed by harsh procedures (which may lessen antigenicity). Vibratome sectioning is an alternative to freezing or embedding and takes advantage of the vibration of an advancing blade to cut tissue blocks immersed in buffer. However, with the vibratome, it is difficult to obtain lung sections thinner than 50-60 μm, which results in incomplete antibody penetration.
Intracellular Double Labeling of Substantia Nigra and Pedunculopontine Neurons in in vitro Slice Preparation
Published in Avital Schurr, Benjamin M. Rigor, BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
For experiments combining intracellular recording and labeling, an animal (i.e., a rat weighing 150–350 g) is decapitated and the brain gently removed from the skull. The brain is placed immediately in a small petri dish containing cold (~5°C) oxygenated Krebs-Ringer’s solution. The brain is cut by hand with a razor blade into an appropriately oriented block containing the structures to be studied (i.e., frontal, sagittal, etc.). For Vibratome™*** sectioning, the tissue block is then glued to the cutting stage of a Vibratome™ using a cyanoacrylic cement. The Vibratome™ bath is filled with cold Krebs-Ringer’s solution, and the block is sectioned at approximately 350–500 μm for intracellular study and 150 to 200 μm for a whole-cell patch-clamp study. Four to six slices are sectioned using a Vibratome™ or a tissue chopper and are stored in a beaker containing continuously oxygenated Krebs-Ringer’s solution at room temperature until each slice is ready to be recorded. We have found it beneficial to store the tissue in this manner for at least 1–2 h prior to recording. Krebs solution is composed of (in mM):NaCl 124; KCl 1.8; KH2PO4 1.24; NaHCO3 26; CaCl22.4; MgSO41.3; and glucose 10.
Fixation and Tissue Pretreatment
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Fixed, unfrozen sections can be cut down to 30 to 40 μm with vibrating-knife instruments. This method has turned out to be extremely useful for the study of numerous neuropeptides and catecholamine-synthesizing enzymes.53,99,100 Some improvements of the sectioning technique have been published by Clouser.22 In order to assure penetration of antibodies and PAP complexes into the rather thick sections, additions of Triton® X-100 to rinsing solutions and antisera solutions have been advocated. The method can be used for pre-embedding immunocytochemical staining. However, penetration is a problem, and when the technique is combined with peroxidase methods, the poor precision of the latter may cause problems for interpreting exactly what subcellular organelles store the immunoreactivity. Piekut and Casey studied 80-μm-thick sections of rat brains and found the staining (PAP method) to penetrate by only 8 to 9 μm.100 Moreover, inclusion of Triton® X-100 in the procedure caused considerable membrane damage. In my own experience, pre-embedding immunocytochemistry of Vibratome® sections is a valid technique for identifying neurons, cell bodies, and processes. However, as the peroxidase-reaction spreads out within the confines of the cell membrane, interpretations of the exact subcellular localization of antigens should be avoided. Use of a particulate tracer such as colloidal gold could conceivably alleviate this problem. However, the poor penetration of such tracers in both Vibratome® and cryostat sections usually necessitates use of such high detergent concentrations that overt ultrastructural damage will ensue. Lamberts and Goldsmith thus recently reported that 16-nm gold particles penetrated only a few microns on the surface of Vibratome® sections.67 More succesful results were obtained by van den Pol.125
Lack of bombesin receptor-activated protein homologous protein impairs hippocampal synaptic plasticity and promotes chronic unpredictable mild stress induced behavioral changes in mice
Published in Stress, 2023
Xueping Yao, Xiaoqun Qin, Hui Wang, Jiaoyun Zheng, Zhi Peng, Jie Wang, Horst Christian Weber, Rujiao Liu, Wenrui Zhang, Ji Zeng, Suhui Zuo, Hui Chen, Yang Xiang, Chi Liu, Huijun Liu, Lang Pan, Xiangping Qu
Mice were sacrificed by intraperitoneal injection of lethal dose of phenobarbital in accordance with the Xiangya animal care and protocols by Central South University. Brain tissues from 6 mice of each group were removed immediately and fixed in at least 20 volumes of 4% paraformaldehyde. Then the tissue preparation and Golgi-Cox staining were conducted following the instructions of FD Rapid GolgiStainTM Kit (FD NeuroTechnologies, PK401). Tissues were cut into 200 µm thick sections with a vibratome. The image of hippocampus was obtained by optical microscopy (BX53, Olympus Corporation). Five neurons were selected from CA1 or CA3 regions of each sample. The apical spines on the secondary and tertiary dendrites of the neurons were counted and the length of spines were measured by ImageJ. The dendritic length and number of the branches were calculated according to Sholl analysis (Sholl, 1953) using ImageJ software with the NeuronJ plugin.
Comparison of tyrosine-modified low molecular weight branched and linear polyethylenimines for siRNA delivery
Published in Nanotoxicology, 2022
Małgorzata Kubczak, Sylwia Michlewska, Michael Karimov, Alexander Ewe, Achim Aigner, Maria Bryszewska, Maksim Ionov
Tumor tissue slices were generated from A549 tumor xenografts. Briefly, 350-µm slices were obtained using Leica VT 1000 vibratome and with a disposable biopsy punch, pieces of 3 mm in diameter were prepared. These punched tissue slice pieces were then placed on membrane culture inserts with 0.4 µm pore size, located in 6-well plates containing 1 ml of RPMI-1640 medium supplemented with 10% FCS and 1% antibiotics, for ail-liquid interface culture. After overnight cultivation under standard cell culture conditions, the slices were transferred into a 96-well plate containing 100 µl pre-warmed medium. Polymer/siRNA complexes were added to the slices for 4 h incubation, then the slices were returned onto the membrane inserts and further cultivated, with the medium being replaced every day. After 72 h, slices were harvested for RT-qPCR analysis.
A Biomechanical and Microstructural Analysis of Bovine and Porcine Pericardium for Use in Bioprosthetic Heart Valves
Published in Structural Heart, 2021
Greg Campion, Kylie Hershberger, Alix Whelan, Jack Conroy, Caitríona Lally, Bruce P. Murphy
For the dog-bone specimens, a 4 mm interrogation region was defined at the center of the gauge length. Three imaging zones, with image depth increments of 10 μm, were recorded in these regions (see Figure 1 for the specific site where these images were acquired). Z-stacks were first recorded on the serous side of the tissue, and then the tissue was repositioned by flipping the tissue 180° about its longitudinal axis to image the fibrous side. Therefore, a total of six z-stacks were recorded for each specimen (three stacks on each of the fibrous and serous surfaces). Z-stacks were recorded until no further signal was detected, or as deep into the tissue as possible. This procedure was followed for imaging the two thinner vibratome delaminated fibrous and serous layers.