Spe 2 confocal microscope

Manufactured by Leica

Sourced in Germany

The SPE-II confocal microscope is a high-performance laboratory instrument designed for advanced imaging applications. It utilizes confocal optics to capture detailed, high-resolution images of microscopic samples. The core function of the SPE-II is to provide researchers and scientists with a versatile, reliable tool for conducting precision-based imaging and analysis tasks.

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30 protocols using spe 2 confocal microscope

C. elegans Fluorescence Imaging

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pfat-7::GFP and SBP-1::GFP C. elegans strains were grown until the L4/young adult transition and images were acquired on a Leica SPE II confocal microscope. All images were taken at identical gain settings within experimental sets and Adobe Photoshop was used for corrections to levels across experimental sets.

Smulan L.J., Ding W., Freinkman E., Gujja S., Edwards Y.J, & Walker A. (2016). Cholesterol-independent SREBP-1 maturation is linked to ARF1 inactivation. Cell reports, 16(1), 9-18.

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Immunofluorescence Staining of ATP7A and Golgin-97 in Cells

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After the indicated treatments outlined in the Results, human fibroblasts or MEF were fixed using 4% (m/v) PFA, for 12 min at room temperature (RT), permeabilized (0.3% (v/v) Triton X-100, 10 min) and blocked (5% (v/v) bovine serum albumin for human cells or 5% (v/v) normal goat serum for MEF, 1 h RT). Cells were incubated with primary antibodies overnight at 4°C and with Alexa Fluor-labeled secondary antibodies (Molecular Probes-Invitrogen, Paisley, UK) at RT for 2 h. Nuclei were counterstained with 300 nM 4,6-diamidino-2- phenylindole (DAPI, Molecular Probes) and mounted using Prolong Gold antifade reagent (Invitrogen). Cells were visualized using a Leica SPE-II confocal microscope and images acquired at 63X magnification. Primary antibodies used include: rabbit monoclonal ATP7A 1:250 (Antibody Solutions) and mouse monoclonal golgin 97 1:500 (CDF4) (Santa Cruz).

Perez-Siles G., Grant A., Ellis M., Ly C., Kidambi A., Khalil M., Llanos R.M., La Fontaine S., Strickland A.V., Züchner S., Bermeo S., Neist E., Brennan-Speranza T.C., Takata R.I., Speck-Martins C.E., Mercer J.F., Nicholson G.A, & Kennerson M.L. (2016). Characterizing the molecular phenotype of an Atp7aT985I conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX). Metallomics : integrated biometal science, 8(9), 981-992.

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Quantifying RGC Survival Post-NMDA Insult

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RGC survival was estimated 3 weeks after NMDA injection on retinal flat-mounts^{2 (link),37 (link),66 (link)}. In brief, flat-mounted retinae were washed with 0.1 M PBS and permeabilized with 100% methanol for 20 min at −20 °C. Retinal flat-mounts were immunofluorescently labeled for β3tubulin, a marker of RGCs. In this aim, retinae were incubated with a rabbit anti-β3tubulin antibody (1:500, Abcam, Cambridge, UK, #ab18207) diluted in blocking solution (PBS containing 5% bovine serum albumin (BSA), 0.3% Triton X-100, and 0.05% sodium azide) and then with a secondary anti-rabbit antibody. Images were taken with a Leica SPE-II confocal microscope at ×40. RGCs were counted in regions of 62 500 µm² at 1 and 1.5 mm from the optic disk in the four retinal quadrants.

Mdzomba J.B., Jordi N., Rodriguez L., Joly S., Bretzner F, & Pernet V. (2018). Nogo-A inactivation improves visual plasticity and recovery after retinal injury. Cell Death & Disease, 9(7), 727.

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Confocal Microscopy Imaging Protocol

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All images were acquired on a Leica SPE II confocal microscope using LAS AF software (Leica) using the 63 × objective and zoomed to 1.7 × the Nyquist limit, followed by deconvolution in Huygens Essential (www.svi.nl) with an estimated PSF and default parameters (except for mounting media refractive index, which was 1.42 per manufacturer’s instructions).

Gilliland W.D., May D.P., Colwell E.M, & Kennison J.A. (2016). A Simplified Strategy for Introducing Genetic Variants into Drosophila Compound Autosome Stocks. G3: Genes|Genomes|Genetics, 6(11), 3749-3755.

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Quantifying ATP7A/Atp7a at the TGN

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For quantification of the ATP7A/Atp7a present at the TGN subsequent to the treatments outlined in the Results, cells were stained with a rabbit monoclonal ATP7A antibody (Antibody Solutions) and a mouse anti-Golgi97 antibody followed by incubation with an anti-rabbit ALEXA-488 and anti-mouse ALEXA-555 secondary antibodies respectively. Using a Leica SPE-II confocal microscope, settings for optimal visualization of ATP7A/Atp7a at the TGN in control samples and basal conditions were determined and used for the acquisition of all subsequent samples in the experiment. At least 20 images were acquired at 63X magnification for each condition. To precisely define the region on each image corresponding to the TGN and specifically quantify the presence of ATP7A/Atp7a at that location, a region of interest (ROI) was defined using the image processing package Fiji^{29 (link)}. Briefly, a threshold was applied to the channel containing the TGN information to set appropriate signal-to-noise ratios. By using the “Analyze Particles” option and selecting an appropriate size for the organelle (5–10 µm), the region corresponding to the TGN was outlined in every channel. Using the multi measure option of the ROI master tool, the mean intensity of the signal corresponding to ATP7A/Atp7a was calculated and averaged for every cell.

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Sperm Cell Immunofluorescence Protocol

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Whole sperm cells and sperm heads were fixed in 4% PFA for 15 min at RT and permeabilized using 0.5% (v/v) Triton X-100 for 15 min, at RT. For acrosin staining, sperm cells were fixed and permeabilized in −20°C methanol for 5 min, mixed with PBS and deposited on slides. After rinsing with PBS, slides were blocked with 1% (w/v) BSA in PBS for 1 h at RT, incubated overnight at 4°C with primary antibodies. Slides were washed again before incubation for 1 h at RT with secondary antibodies or 20 min with peanut agglutinin lectin (PNA) (Supplementary Table S1) and a Hoechst 33342 counterstain added for 10 min, at RT (1 μg/ml; Sigma). After extensive washing with PBS, slides were mounted with FluorSave reagent (Merck Millipore) and covered with coverslips. For all negative controls the primary antibody was omitted. Images were taken on a Leica SPE-II confocal microscope using a ×63 objective (NA 1.3, HCX PLANAPO oil) and images were analyzed using ImageJ software (bundled with 64-bit Java 1.8.0_172; National Institutes of Health, Bethesda, MD, United States).

Zhang M., Chiozzi R.Z., Skerrett-Byrne D.A., Veenendaal T., Klumperman J., Heck A.J., Nixon B., Helms J.B., Gadella B.M, & Bromfield E.G. (2022). High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins. Frontiers in Cell and Developmental Biology, 10, 836208.

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Immunostaining of transfected HeLa cells

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HeLa cells grown on coverslips in 24-well plates were transfected with 400 ng of the plasmid DNA using Lipofectamine2000 (Thermo Fisher Scientific). At 16 hours post transfection, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and immunostained with the appropriate primary and secondary antibodies diluted in 2% normal goat serum in PBS. Sources of the antibodies were as follows: anti-ACBD3 (Sigma, WH0064746M1), anti-PI4KB (Merck, 06–578), anti-GM130 (BD Biosciences, 610822), anti-giantin (Enzo Life Science, ALX-804-600-C100), anti-myc (Thermo Fisher Scientific, PA1-981), anti-PI4P (Echelon, Z-P004), and goat-anti-mouse and goat-anti-rabbit secondary antibodies conjugated to AlexaFluor 488, 596, or 647 (Molecular Probes). Nuclei were stained with DAPI. Coverslips were mounted with FluorSave (Calbiochem), and confocal imaging was performed with a Leica SpeII confocal microscope.

Horova V., Lyoo H., Różycki B., Chalupska D., Smola M., Humpolickova J., Strating J.R., van Kuppeveld F.J., Boura E, & Klima M. (2019). Convergent evolution in the mechanisms of ACBD3 recruitment to picornavirus replication sites. PLoS Pathogens, 15(8), e1007962.

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Immunofluorescence and Immunohistochemistry of Colorectal Cancer Samples

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Samples undergoing immunofluorescence were either FFPE tumor specimens or growing agnospheres or colosphere mCRC729. The latter were harvested, fixed 10 min with PFA 4% at 4 °C, washed in PBS, suspended in bio-agar for cyto-inclusion (Bio-Optica) at 42 °C, and processed for inclusion in paraffin. All staining were performed as previously described^{67 (link)}. Images were acquired using a LEICA SPEII confocal microscope, equipped with a 40× oil objective and a 1.5× zoom for a final magnification of 600×. Optical single sections were acquired with a scanning mode format of 1024 × 1024 pixels. Fluorochrome unmixing was performed by acquisition of automated-sequential collection of multichannel images, to reduce spectral crosstalk between channels. For immunohistochemical staining, an additional peroxidase blocking was performed in H₂O₂ 3% methanol 50% incubated 20 min in the dark. For primary and secondary antibody concentrations, see reagents. Secondary antibodies were HRP-conjugated (Dako), and DAB substrate chromogen kit (Dako) was used for detection. Nuclei were counterstained with hematoxylin. Images were acquired through LASV4.2 software and are representative of at least three independent immunostainings.

Verginelli F., Pisacane A., Gambardella G., D’Ambrosio A., Candiello E., Ferrio M., Panero M., Casorzo L., Benvenuti S., Cascardi E., Senetta R., Geuna E., Ballabio A., Montemurro F., Sapino A., Comoglio P.M, & Boccaccio C. (2021). Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition. Nature Communications, 12, 2498.

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Immunofluorescence Staining of HT29 Cells

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HT29-MTX and HT29-MTX ∆MUC1 cells were grown for 7 d to reach a confluent monolayer on coverslips (8 mm diameter #1.5) in 24-well plates. Cells were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS, D8537) and fixed with 4% paraformaldehyde in PBS (Affymetrix) for 30 min at RT. Fixation was stopped by adding 50 mM NH₄Cl in PBS for 15 min. Cells were washed two times and permeabilized in binding buffer (0.1% saponin [Sigma-Aldrich] and 0.2% BSA [Sigma-Aldrich] in DPBS) for 30 min. Coverslips were incubated with 139H2 WT, synthetic FAB at 1:100 dilution for 1 h, washed 3× with binding buffer, and incubated with Alexa Fluor 488–conjugated α-mouse IgG secondary antibodies (1:200; A11029; Thermo Fisher Scientific) and DAPI at 2 μg/ml (D21490; Invitrogen) for 1 h. Coverslips were washed 3× with DPBS, desalted in Milli-Q, dried and embedded in ProLong Diamond Mounting Solution (Thermo Fisher Scientific), and allowed to harden. Images were collected on a Leica SPE-II confocal microscope with a 63× objective (NA 1.3, HCX PLANAPO oil) and controlled by Leica LAS AF software with default settings to detect DAPI, Alexa Fluor 488, Alexa Fluor 568, and Alexa Fluor 647. Axial series were collected with step sizes of 0.29 μm.

Peng W., Giesbers K.C., Šiborová M., Beugelink J.W., Pronker M.F., Schulte D., Hilkens J., Janssen B.J., Strijbis K, & Snijder J. (2024). Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry–based de novo sequencing. Life Science Alliance, 7(6), e202302366.

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RNA Expression Analysis in hCSs

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hCSs were processed as described for IHC. RNAScope was performed according to the manufacturer’s protocol, with antigen retrieval (ACDBio). Human probes were custom-made by ACDBio for PAX6, TUBB3, and HES1 mRNA. After RNAScope was performed, sections were immediately imaged or stored dried overnight in the dark at 4°C and imaged the following day. Imaging was performed using a Leica SPE-II confocal microscope at 20× tile scan. One section from three independent hCSs were imaged and analyzed. Quantification was performed using CellProfiler, modified for use. Data were reported as the ratio of target mRNA to DAPI expression.

Hurley E.M., Mozolewski P., Dobrowolski R, & Hsieh J. (2023). Familial Alzheimer’s disease-associated PSEN1 mutations affect neurodevelopment through increased Notch signaling. Stem Cell Reports, 18(7), 1516-1533.

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