Formalin-fixed, paraffin embedded (FFPE) tissue blocks containing the hippocampus that were collected and processed as part of routine autopsy procedures were used in this study, which were matched in the rpAD and sAD cases. A subset of these tissue blocks were treated with 98% formic acid prior to paraffin embedding because of the presence of suspected prion disease. Formic acid treated tissue blocks were immersed in 98% formic acid for 1 hour at room temperature, rinsed 3 times with 10% formalin and allowed to post-fix with 10% formalin for an additional 48 hours prior to standard paraffin embedding. Immunostaining for amyloid plaques and laser capture microdissection (LCM) was performed using the method detailed in Drummond et al.[23 (link)]. Briefly, 8μm sections of FFPE tissue blocks were collected onto LCM-compatible slides and amyloid plaques were visualized using fluorescent immunohistochemistry using a combination of the pan-Aβ antibodies 4G8 (1:4000; BioLegend; Catalog #800709) and 6E10 (1:4000; BioLegend; Catalog #803001). LCM was performed using a LMD 6500 microscope (Leica). 2mm2 total area of fluorescently-labeled plaques was manually selected, microdissected using LCM and collected into double distilled water. Samples were stored at −80°C until sample processing for LC-MS.
Lmd6500 microscope
Manufactured by Leica
The LMD6500 is a laser microdissection microscope designed for precise tissue sample extraction. It features high-resolution optics, a motorized stage, and a laser beam for targeted dissection of small tissue regions. The core function of the LMD6500 is to allow users to isolate specific cells or regions of interest from heterogeneous samples for downstream analysis.
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Lab products found in correlation
5 protocols using lmd6500 microscope
1
Laser Capture Microdissection of Amyloid Plaques
2
Laser Capture Microdissection of Temporal Cortex in Advanced Alzheimer's Disease
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LCM was performed using a LMD6500 microscope (Leica) on sections of temporal cortex collected from two patients with advanced AD. Informed consent for autopsy was obtained ante-mortem from subjects and post-mortem from next of kin in accordance with guidelines from the Institutional Review Board (IRB) of NYU Langone Medical Center (NYULMC). All experimental protocols were approved by the NYULMC IRB. Assessment of pathology was performed by a neuropathologist (TW) and AD pathology was graded using the “ABC” criteria, as recommended by the National Institute on Aging-Alzheimer’s Association guidelines44 (link). Both patients had advanced AD pathology with ABC scores of A3B3C3. Patient characteristics are outlined in Table 2 . Blocks of tissue from each of the patients were formalin fixed and paraffin embedded as part of routine autopsy procedures. 8 μm thick sections containing the temporal cortex were cut using a microtome and collected onto LCM compatible polyethylene terephthalate (PET) frame slides (Leica).
3
Laser-Capture Microdissection of Brain Tissue
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After fixation in formalin, FFPE brain tissue blocks containing either the superior frontal gyrus of the frontal cortex or hippocampus (level of LGN) were sectioned at 8 µm and collected onto LCM compatible PET slides (Leica). Sections were stained with cresyl violet to localize regions of interest for LCM [15 (link)] and air dried overnight in a loosely closed container. LCM was used to individually microdissect 10 mm2 from the frontal cortex (gray matter layers I–IV) and hippocampal CA1-3, and 4 mm2 from the hippocampal DG into LC–MS grade water (Thermo Scientific). Microdissected samples were centrifuged for 2 min at 14,000 g and stored at – 80 °C. LCM was performed at 5X magnification with a LMD6500 microscope equipped with a UV laser (Leica).
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Sectioning and Microdissection of FFPE Brain Tissue
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FFPE brain tissue blocks containing either hippocampus (lateral geniculate nucleus level)19 (link) or superior frontal gyrus were sectioned at 8 µm and collected onto laser capture microdissection (LCM)–compatible PET slides (Leica, Newcastle, UK). Sections were stained with cresyl violet to localize regions of interest for LCM20 (link) and air dried overnight in a loosely closed container. LCM was used to individually microdissect 10 mm2 from the hippocampal CA1-3 region and superior frontal cortex (layers I–IV), and 4 mm2 from the hippocampal dentate gyrus into liquid chromatography (LC)–mass spectrometry (MS)–grade water (Thermo Scientific, Waltham, MA). Microdissected samples were centrifuged for 2 minutes at 14,000g and stored at −80°C. LCM was performed at 5× magnification with an LMD6500 microscope equipped with an ultraviolet laser (Leica).
5
Laser Capture Microdissection of FFPE Brain Tissue
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We sampled brain tissue from formalin-fixed paraffin embedded (FFPE) blocks of hippocampus or superior frontal gyrus collected and processed for autopsy neuropathology. LCM was performed using our published protocol [31] [32] [33] . Briefly, 8µm-thick FFPE sections containing hippocampus or frontal superior gyrus were collected onto LCM PET Frame Slides (Leica). Sections were stained with cresyl violet to visualize neurons and regions of interest (ROIs). For each sample, 10mm 2 of hippocampus (including CA1-3) and superior frontal gyrus (including layers I-IV) and 4mm 2 of dentate gyrus (granule cell layer) were microdissected in separate tubes using a LMD6500 microscope at 5X magnification (Leica) (Figure 1). ROIs were collected into ultrapure Pierce Water, LC-MS Grade (Thermo Scientific).
After collection, samples were centrifuged at 14,000g for 2min and stored at -80˚C until peptide extraction.
After collection, samples were centrifuged at 14,000g for 2min and stored at -80˚C until peptide extraction.
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