Paraffin sections were deparaffinized in xylene, hydrated in a graded series of ethanol, and blocked by incubation with 1% BSA in PBS for 2 h. The following primary antibodies were used at 4 °C overnight at the indicated dilutions: α-SMA (1:100; Biolegend); IL-6 (1:30; Invitrogen); Ki67 (SP6; 1:200; Abcam); pan-cytokeratin (AE1/AE3 + 5D3; 1:200; Abcam); E-cadherin (1:200; R&D Systems, Wiesbaden, Germany); N-cadherin (1:200; BD Transduction Laboratories, Franklin Lakes, NJ, USA); and collagen type 1 (1:200; Invitrogen). After washing with PBS, sections were incubated for 2 h at room temperature in secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:400; Alexa Fluor 488 goat anti-rabbit IgG, 1:400; Alexa Fluor 488 Donkey anti-Goat IgG, 1:400; Alexa Fluor 594 goat anti-mouse IgG, 1:400; and Alexa Fluor 594 goat anti-rabbit IgG, 1:400). All secondary antibodies were purchased from Invitrogen. Phase-contrast images were acquired on a Zeiss LSM 880 inverted Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany).
Lsm 880 inverted confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM 880 is an inverted confocal laser scanning microscope. It is designed to capture high-resolution images of samples by using a laser to scan the specimen and detect the resulting fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
α sma, by BioLegend (1 mentions)
Ki67 sp6, by Abcam (1 mentions)
N cadherin, by BD (1 mentions)
Sytox orange dead cell stain, by Thermo Fisher Scientific (1 mentions)
Wga alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Imaris software, by Oxford Instruments (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
3 protocols using lsm 880 inverted confocal laser scanning microscope
1
Immunofluorescence Staining of Paraffin Sections
2
Biofilm Staining and Microscopy Protocol
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The biofilm model was gently washed by adding 500 μl milli‐Q water. The gel was then removed from its mould, placed on a microscope slide and left to dry for 1 h. A mix of Syto™9, Sytox™Orange Dead Cell Stain and wheat germ agglutinin (WGA) Alexa Fluor® 594 conjugate (Invitrogen) was prepared and added to the gel. Sytox™Orange was used to indicate permeable membranes. Therefore, cells which appear yellow or orange in colour have permeable membranes and are assumed to be dead or dying. The use of WGA, a lectin binding specifically to the N‐acetylglucosamines in the peptidoglycan layer of bacterial cell walls, served as a morphology‐independent marker to distinguish Gram‐negative PA 14 from Gram‐positive S. aureus.31 (link) With these stains, live P. aeruginosa appeared green, dead P. aeruginosa appeared orange, live S. aureus appeared green with a slight red “halo” around it, and dead S. aureus appeared yellow with a red halo. The stained gel was left to incubate in the dark for 15 min. A razor blade was used to cut thin vertical slices of 2–3 mm thickness from the gel. These were visualized in a Zeiss LSM 880 inverted confocal laser scanning microscope (Carl Zeiss GmbH, Germany), and the images were subsequently processed using the IMARIS software (Bitplane AG, Switzerland).
3
Examining Sec31A Morphology in ALS
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Five micrometer paraffin-embedded, formalin-fixed human post-mortem cervical spinal cord tissue sections from three neurologically normal controls and three ALS patients with C9orf72 repeat expansions (obtained from the NSW Brain Tissue Resource Centre, collected at autopsy) were used to examine Sec31A morphology. Patient demographic information is detailed in Table 1. Fluorescent immunohistochemistry was performed as previously described [28] (link). Briefly, tissue sections were dewaxed and rehydrated in xylene and a graded ethanol series of decreasing concentrations. Antigen retrieval was performed by boiling tissues in citric acid buffer at 100 °C for 30 min. Tissues were then immunolabelled with primary anti-Sec31A antibody (1:100, Sigma HPA005457), followed by AlexaFluor 555 conjugated secondary antibody (1:250, Invitrogen). Nuclei were counterstained with DAPI (1:5,000, Invitrogen) and tissues were mounted with fluorescent mounting medium (Dako). At least five neurons per case were imaged using a Zeiss LSM 880 inverted confocal laser scanning microscope (Zeiss) at 100 × /na = 1.46 magnification. Neurons in Sec31A-channel images were selected using the freehand selection tool on Fiji Image J, excluding lipofuscin. This work was approved by the Macquarie University Human Ethics Committee (Ref: 5201600719).
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