Three AAmut and three AOD samples were collected for polychromatic immunofluorescence staining. Four percent formalin was used to fix the AG specimens and the specimens were embedded in paraffin blocks. For polychromatic immunofluorescence, 3 µm paraffin sections were washed in PBS twice, and permeabilized in 0.2 to 0.5% Triton X-100 (Solarbio). Then the paraffin sections were blocked in 5% normal donkey serum (Jackson Lab) and stained with primary antibody. Fluorescent-conjugated secondary antibodies (ZSGB-Bio) were used to detect the primary antibodies. Fluorescence mounting medium (Dako) was used to mount the sections. As previously described (18 (link)), we used the Opal 4-Color Manual IHC Kit (Perkin Elmer) for the analysis of formalin-fixed paraffin-embedded AG sections following the manufacturer’s protocols. Zeiss LSM880 NLO microscope was used to acquire fluorescent images. Primary antibodies were anti-CD45 (OriGene), anti-lba1 (CST), and anti-CD206 (Proteintech). GAMs were defined based on cells that costained with CD45 and lba1. CD206+ GAMs were defined based on cells that were costained with CD45, lba1, and CD206. The percentage of CD206+ GAMs was defined by (CD206+ GAMs)/GAMs.
Lsm 880 nlo microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM 880 NLO is a multiphoton microscope designed for advanced imaging applications. It features a high-performance laser system and a sophisticated optical design to enable high-resolution, deep-tissue imaging. The microscope is optimized for nonlinear optical techniques, including two-photon excitation fluorescence and harmonic generation imaging.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Solarbio (3 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (3 mentions)
Fluorescence mounting medium, by Agilent Technologies (2 mentions)
Opal 4 color manual ihc kit, by PerkinElmer (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Anti cd206, by Proteintech (1 mentions)
Axiophot light microscope, by Zeiss (1 mentions)
Ab134933, by Abcam (1 mentions)
Anti cd86, by Abcam (1 mentions)
Axio scope al, by Zeiss (1 mentions)
Anti cd68, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
T9026, by Merck Group (1 mentions)
L9393, by Merck Group (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Donkey anti goat alexa 647, by Jackson ImmunoResearch (1 mentions)
Goat anti iba 1, by Novus Biologicals (1 mentions)
Donkey anti mouse alexa 488, by Jackson ImmunoResearch (1 mentions)
11 protocols using lsm 880 nlo microscope
1
Polychromatic Immunofluorescence Staining of GAMs
2
Immunofluorescence Analysis of Perforin and Granzyme B in CRC and NK92-MI Cells
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CRC tissues and NK92-MI cells were fixed in 4% formalin at 4 °C for 8 h, embedded in paraffin blocks, and cut into 3-µm sections, which were mounted onto slides. The slides were then permeabilized in 0.2–0.5% Triton X-100 and blocked in 5% normal donkey serum at room temperature for 1 h. The slides were incubated with anti-perforin (1:250, Santa Cruz, sc-373,943) and anti-granzyme B (1:250, Abcam, ab134933) antibodies overnight and then incubated with DAPI and fluorescence-conjugated goat anti-mouse IgG H&L (1:500, Abcam, ab150117). Finally, the slides were fixed with fluorescence mounting medium (Sangon) and imaged using a Zeiss LSM880 NLO microscope. The CRC tissues were probed with an anti-NK1.1 monoclonal antibody (Thermo Fisher #16-5941) and examined using an Axiophot light microscope (Zeiss, Oberkochen, Germany).
3
Immunofluorescence Analysis of Atherosclerotic Plaques
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Plaques from 9 patients (4 vulnerable plaques and 5 stable plaques) were fixed overnight in 4% formalin (4°C) and embedded in paraffin blocks for paraffin sections. Three paraffin sections (4 um) were cut from each specimen. Hematoxylin and eosin (H&E) staining was performed. For immunofluorescence, paraffin sections were washed twice 15 min in PBS (Sigma-Aldrich, United States), permeabilized in 0.2%-0.5% Triton X-100 (Solarbio, China) and blocked in 5% normal donkey serum (Jackson Lab, United States) for 1 h and stained with primary antibody overnight. Primary antibody was detected using fluorescent-conjected second antibodies (ZSGB-BIO, China). Primary antibodies were: anti-CD68 (Abcam, United States), anti-CD86 (Abcam, United States), and anti-HLA_DR (Abcam, United States). Sections were mounted with fluorescence mounting medium (Glostrup, Denmark). Fluorescent images were acquired on a Zeiss LSM880 NLO microscope and Zeiss Axio Scope Al was used to obtain H&E images. Three fields were randomly selected from each staining and the number of fusion particles of CD68, CD86 and HLA_DR in each field was counted. The Wilcoxon rank test was used and P<0.05 indicated statistically significant difference.
4
Immunohistochemical Analysis of Embryo Tissues
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Five healing embryos per condition were fixed in PFA overnight, washed three times in PBT and mounted into 4% agarose diluted in water. Next, 150 μm sections were prepared using Leica Vibratome (VT1000 S), and immunohistochemistry was performed according to Sive et al. [123 ] protocol using primary antibodies against β-catenin (1:1000, Sigma-Aldrich T9026), laminin (1:150, Sigma-Aldrich L9393) and actin - Alexa Fluor 488 phalloidin (1:1000, Life Technologies A12379). The secondary antibody Alexa 488 goat anti-rabbit (1:500, Life technologies A11008) was used for laminin and β-catenin staining. Samples were imaged using Carl Zeiss LSM 880 NLO microscope and images were processed using Zen (Zeiss) and Zoner Photo Studio 17 software.
5
Upconversion Nanoparticle Imaging Protocol
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To visualize the UCNPs, a Carl Zeiss LSM 880 NLO microscope (Oberkochen, Germany), equipped with a 40× NA1.1 water immersion objective and a 32 GaAsP array spectral detector covering emission from 410 to 694 nm and operated at single photon counting mode for maximum SNR, was used. Lambda mode at full spectral resolution was used to spectrally prove UCNPs emission (two characteristic distinct and narrow peaks at 544 nm and 660 nm) and a channel mode was used to combine DAPI (405 nm excitation, 410–500 nm emission bands), UCNPs (974 nm excitation, 535–565 nm and 650–668 nm emission bands), and transmitted light signals. A 974 nm excitation by a TiSa laser (80 MHz, 350 fs laser pulse width at sample plane) provided the highest emission intensity for upconversion and was used for imaging. The 974 nm laser power was kept low at <50 µW at the sample plane. To capture the slow emission of UCNPs (excited state lifetime on the order of hundreds of microseconds), the scanning speed was the slowest possible, 132 µs per pixel, and the pinhole was opened to 300 um (around 4 Airy units). The pixel size was 132 nm, the image size was 512 × 512 pixels, and bidirectional scanning was used.
6
Perfusion and Immunostaining of Trigeminal Ganglia
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Five days after surgery, mice were intracardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) and the TGs were removed. TGs were left in PFA at 4°C for 6 hours. For staining with an anti-GFP antibody (table S4), the ganglia were pretreated with 100 μM digitonin according to a previously established protocol to preserve DiI from diffusion (57 (link)). The ganglia were mounted in agarose, and image stacks were acquired with a Zeiss LSM 880 NLO microscope equipped with a 680- to 1300-nm tunable and fixed 1040-nm two-photon laser (Newport Spectra-Physics) and a 20× W-Plan Apochromat objective lens. Fluorophores were excited at 1040 nm (DiI) and 920 nm (tau GFP), and fluorophore emissions were detected with nondescanned GaAsP detectors at 575 to 610 nm (DiI) and 500 to 550 nm (tau GFP). Fiji ImageJ was used to process the data, and the ImageJ Cell Counter plug-in was used to quantify cell numbers. For TGs, two different experimenters counted the cell bodies; the results represent their mean score.
7
Immunohistochemical Profiling of Microglia
8
Immunofluorescence Microscopy of SMCHD1
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Immunofluorescence microscopy studies were performed as described previously [37 (link)]. Briefly, cells were washed in PBS and fixed in 3% (w/v) paraformaldehyde made in PBS for 10 min at room temperature. Cells were washed three times with PBS for 5 min each, then permeabilized on ice with 0.5% (v/v) Triton X-100 in PBS, followed by three washes in PBS for 5 min each. Cells were blocked in 1% (w/v) bovine serum albumin (BSA) (Life Technologies) for 15 min, followed by an overnight incubation in a dark and humid chamber at 4°C with a primary anti-SMCHD1 antibody (in-house clone 2B8; available from Millipore under catalog number MABS2292) diluted 1 : 100 in 1% (w/v) BSA. Cells were washed three times in PBS for 5 min each and incubated for 40 min at room temperature in a dark and humid chamber with a secondary anti-rat-568 antibody for SMCHD1 (Life Technologies, A-11077) diluted 1 : 500 in 1% (w/v) BSA. Cells were washed three times in PBS for 5 min each and stained with DAPI for 10 min at room temperature, followed by another two PBS washes. Coverslips were mounted in Vectashield H1000 mounting medium (Vector Laboratories). Cells were visualized using the Zeiss LSM 880 NLO microscope at 63× magnification and z-stacks were acquired. Images were analyzed using the open source ImageJ distribution package, FIJI.
9
Immunohistochemical Analysis of Dopaminergic Neurons
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DA samples were fixed overnight at 4°C in 4% formalin and embedded in paraffin blocks to obtain paraffin sections. Immunohistochemical staining was performed as previously reported (25 (link)). For immunofluorescence, 3 μm paraffin sections were washed twice in PBS (catalog no. 806544, Sigma-Aldrich) for 15 min, permeabilized in 0.2–0.5% Triton X-100 (catalog no. T8200-100, Solarbio) and blocked in 5% normal donkey serum (catalog no. 017-000-001, Jackson Lab) for 1 h and stained with primary antibody overnight. The primary antibodies were detected using fluorescent-conjugated secondary antibodies (catalog no. PV-6000, ZSGB-BIO). Sections were mounted with fluorescence mounting medium (catalog no. S3023, Dako). As previously reported (26 (link)), the Opal 4-Color Manual IHC Kit (catalog no. NEL810001KT, Perkin Elmer) was used for the analysis of the formalin-fixed paraffin-embedded DA sections according to the manufacturer's protocol. Fluorescent images were acquired with a Zeiss LSM880 NLO microscope. The primary antibodies were anti-CD45 (catalog no. AB40763, Abcam), anti-CD11b (catalog no. 21851-1-AP, Proteintech), anti-TNFα (catalog no. 60291-1-Ig, Proteintech), and anti-IDO (catalog no. 86630S, CST).
10
Circular RNA and miRNA FISH Analysis
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Specific probes targeting circ_002117 and miR-370 were used for FISH analysis. A Cy5-labeled probe recognized circ_002117 while the farm-labeled probe recognized miR-370. The nucleus was stained by DAPI. All FISH procedures were in accordance with the kit manufacturer’s instructions (Genepharma, China). Images were obtained using a Zeiss LSM880 NLO microscope (2 + 1 with BIG).
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