Whole-mount samples were stained and cleared with a modified 3DISCO protocol37 (link). In short, samples stored in PBS-GT were incubated with primary antibodies in PBS-GT with shaking, for 36 h at RT. Excessive antibody was removed by thorough washing in PBS-GT for 6–12 h and refreshing the solution every 1–2 h. Incubation with fluorophore-coupled secondary antibodies (Molecular Probes) in PBS-GT for 36 h was followed by thorough washing in PBS-GT as described above. When necessary, samples were dehydrated in an ascending Tetrahydrofuran (Sigma, #186562) series (50%, 70%, 3 × 100%; 60 min each), and subsequently cleared in dichloromethane (Sigma, #270997) for 30 min and eventually immersed in benzyl-ether (Sigma, #108014). Non-cleared samples were imaged in 35 mm glass-bottom dishes (Ibidi, #81218) using a laser scanning confocal microscope (Zeiss LSM710) or SP8 Multiphoton microscope (Leica). Cleared samples were imaged whilst submerged in benzyl-ether with a light-sheet fluorescence microscope (LaVision BioTec).
Sp8 multiphoton microscope
Manufactured by Leica
Sourced in Germany
The Leica SP8 multiphoton microscope is a state-of-the-art imaging system designed for advanced live-cell and tissue imaging. It utilizes multiphoton excitation technology to capture high-resolution, deep-tissue images with minimal photodamage. The SP8 is capable of performing multi-label fluorescence imaging, enabling the simultaneous visualization of multiple cellular structures and processes.
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Lab products found in correlation
Lsm710, by Leica (1 mentions)
Light sheet fluorescence microscope, by Miltenyi Biotec (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Application suite x, by Leica (1 mentions)
Application suite x software, by Leica (1 mentions)
Huygens professional version 18, by Scientific Volume Imaging (1 mentions)
Vivaspin filter, by Sartorius (1 mentions)
Sea block, by Thermo Fisher Scientific (1 mentions)
Ab152, by Merck Group (1 mentions)
N4142, by Merck Group (1 mentions)
Synaptopodin, by Santa Cruz Biotechnology (1 mentions)
Sc8298, by Santa Cruz Biotechnology (1 mentions)
Sc21537, by Santa Cruz Biotechnology (1 mentions)
35 mm dish, by BD (1 mentions)
Las x, by Leica (1 mentions)
Nusieve gtg agarose, by Lonza (1 mentions)
12 protocols using sp8 multiphoton microscope
1
3DISCO Whole-Mount Sample Staining
2
In-Situ Redox Imaging of Live Muscle
3
In Vivo Multiphoton Imaging of Microglia
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For acute or chronic imaging, mice were anesthetized with isoflurane (5% for induction; 1–2% for maintenance). The head of the anesthetized mice was stabilized and mounted by the head plate and the animal was placed on a heating plate at ~35 °C under the two-photon microscope. One hundred microliters of Rhodamine B dye (2 mg/mL) was injected intraperitoneally or subcutaneously to label the vasculature. For longitudinal imaging, the blood vessel architecture visible through the craniotomy window was carefully recorded as a precise map of the brain region being visualized and was used to trace back to the original imaging site for chronic imaging studies60 . Imaging was conducted using a Leica SP8 Multiphoton microscope with a coherent laser. A wavelength of 880 nm was optimal for imaging both microglia and the blood vessel dye. The power output at the brain was maintained at 25 mW or below. Images were collected at a 1024 × 1024 pixel resolution using a 25 × 0.9 NA objective with a 1.5× optical zoom. Several fields of view of z-stack images were collected every 1–2 μm through a volume of tissue and used for analysis. To observe microglial dynamics, z-stack time-lapse images were acquired every minute at 2 μm steps in depth. For laser injury, the laser’s power was adjusted to 250 mW for 1 s at 880 nm wavelength and ×48 magnification.
4
Immunostaining of Lymph Node Cryosections
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LN cryosections are stained using a standard protocol. Frozen sections are blocked with 5% mouse serum for 1 hour. Samples were then incubated overnight at 4°C with primary antibodies. After three washes in PBS, samples were incubated with fluorescent probe-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 hour. The primary antibodies used are rabbit anti-Lyve-1 (Clone aa24-228), purified rat anti-mouse CD169 (Clone 3D6.112), anti-Collagen I antibody (Clone EPR22894-89), purified anti-mouse/human GL7 antigen (Clone GL7), Lectin from Arachis hypogaea (PNA) (SIGMA, L6135-1 MG), purified rat anti-mouse CD45R (Clone RA3-6B2), purified rat anti-mouse CD21/35 (Clone 7E9), purified rabbit anti-mouse Ki67 (Clone SP6), purified rabbit anti-mouse elastin (Clone EPR20603).
Slides were imaged using an SP8 confocal microscope (Leica). The objectives used were 20× air and 63× oil, depending on the study. LN sections were collected at the center area of the LNs to ensure MS, SCS, and LN parenchyma were representative. Whole mount lymph node samples were imaged using an SP8 multiphoton microscope (Leica). The objective used was 25× water.
Slides were imaged using an SP8 confocal microscope (Leica). The objectives used were 20× air and 63× oil, depending on the study. LN sections were collected at the center area of the LNs to ensure MS, SCS, and LN parenchyma were representative. Whole mount lymph node samples were imaged using an SP8 multiphoton microscope (Leica). The objective used was 25× water.
5
Macro-Confocal and Multi-Photon Imaging of Cartilage Explants
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Macro-confocal images were obtained with the Leica Macro-confocal TCS LSI at 1× magnification. Images were captured in a format of 1024 × 1024 pixels with a scanning speed of 600 Hz and z-stack steps of 10 µm. CMFDA was visualised using a 488 nm laser line; PI was visualised under sequential scanning using the 532 nm laser line.
All other confocal images were captured using an upright Leica SP8 multi-photon microscope. Explants were visualised using a 10× objective, with the superficial zone oriented towards the objective in a PBS-lined 30 mm plate. A 1024 × 1024 pixel image size was collected for each explant under a scanning speed of 700 Hz with a step size of 2.5 µm. CMFDA and PI were visualised simultaneously with a laser emission bandwidth of 484–555 nm. The gain of the 488 ATOF laser was adjusted to 10.0 as compensation between the two dyes. To avoid operator bias and to ensure the whole explant was represented, a mark-and-find protocol was used to define 5 fields of view for analysis over the explant, which was kept consistent for each explant during one experiment. Total z-stack range varied between images due to the natural curvature of the explant.
All other confocal images were captured using an upright Leica SP8 multi-photon microscope. Explants were visualised using a 10× objective, with the superficial zone oriented towards the objective in a PBS-lined 30 mm plate. A 1024 × 1024 pixel image size was collected for each explant under a scanning speed of 700 Hz with a step size of 2.5 µm. CMFDA and PI were visualised simultaneously with a laser emission bandwidth of 484–555 nm. The gain of the 488 ATOF laser was adjusted to 10.0 as compensation between the two dyes. To avoid operator bias and to ensure the whole explant was represented, a mark-and-find protocol was used to define 5 fields of view for analysis over the explant, which was kept consistent for each explant during one experiment. Total z-stack range varied between images due to the natural curvature of the explant.
6
Quantifying Microglial-Capillary Changes
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Two-photon images were collected using the Leica SP8 Multi-photon microscope with the Leica Application Suite X version 3.5.7.23225 software. Confocal images were collected with a Leica TCS SP8 confocal microscope using the Leica Application Suite X version 3.5.5.19976 software. For blood vessel diameter analysis, two-photon images were collected of microglia and capillaries every other day (days 0, 2, and 4) from either control or PLX3397-treated mice. From collected images, capillaries were selected at random, and their lengths were measured in both conditions on the first (day 0) and fifth day (day 4) of control or PLX3397 treatment. The percent change in the capillary size was determined as a ratio of the capillary size by the fifth day compared to the first day of control or PLX3397 treatment. At least five capillaries from three to five fields of view (11 fields of view from 3 control and 13 fields of view from 3 PLX3397-treated mice) were quantified.
7
Quantifying Collagen in Tissue Samples
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Images were acquired on a Leica SP8 multi-photon microscope equipped with a Mai Tai DeepSee laser tuned to 930 nm. Second harmonic generation signal was collected between 455–475 nm using a HyD detector in photon counting mode and the autofluorescence was collected from 480–600 nm. Channel separation and stitching were performed within the Leica Application Suite X software (Leica Microsystems, Wetzlar, Germany) controlling the microscope, before being deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, Hilversum, The Netherlands). The data was 3D median filtered to remove noise, the background subtracted, and the amount of collagen quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). A Fast Fourier Transform (FFT) filter was applied to the autofluorescence channel to remove stitching artifacts. Image projections are summed slices of the 3D z stack, and to enable visualisation of the total amount of collagen, spanning the entire dynamic range in a single image, a gamma correction of 0.2 was applied.
8
Labeling and Tracking Exosome Uptake
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Freshly isolated exosomes were labeled with the PKH26 red fluorescent cell marker kit (Sigma Aldrich; St. Louis, MO) according to the manufactures instructions and the following modifications. In the final isolation step the exosomes were resuspended in PBS and further mixed with diluent C and the red fluorescent lipophilic dye, PKH26, supplied by the manufacturer. The excess dye was removed by adding 1% BSA solution. The samples were transferred to 30,000 MW vivaspin filters (Sartorius Stedim North America) and washed three times. The samples were transferred to new vivaspin filters and washed with 5 ml of cell culture media. The final exosome pellet was reconstituted in 2 ml of serum free media before adding it to cells. Exosomes were incubated on the cells for one hour and after one hour the exosomes were removed from the cells by washing the cells three times with PBS. The cells were then incubated with 4% paraformaldehyde solution for 15 min and washed three more times. The membrane was then mounted on a glass slide with mounting medium containing DAPI. The slides were then subject to analysis by confocal microscopy. Microscopy images were obtained using a Leica SP8 multiphoton microscope using an HC PL APO 63X 1.40 NA oil objective.
9
Three-Dimensional Imaging of Kidney Slices
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Three-dimensional imaging was performed as previously described (Lindström et al., 2018) by carrying out whole-mount immunofluorescence stains on slices of MD-GFP mouse kidneys.
Slices were fixed in 4% formaldehyde in 1x phosphate buffer saline (PBS) at room temperature (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 7, 2021. ; https://doi.org/10.1101/2021.12.06.471478 doi: bioRxiv preprint for 45 min, washed in 1XPBS, blocked in 1xPBS with 0.1% TritonX100 and 2% SEA Block (ThermoFisher Scientific) for 1 hour, and sequentially incubated in primary and secondary antibodies over 2 days. Primary antibodies were as follows: tyrosine-hydroxylase (AB152, MilliporeSigma, 1:100), GFP (ThermoFisher, 1:200). To clear tissue slices, the slices were dehydrated in methanol via increasing concentrations 50%, 75%, 100% diluted in PBS -each for 1hr -and subsequently submerged in a 50:50 benzyl benzoate/benzyl alcohol (BABB): methanol solution, followed by 100% BABB. High resolution imaging of MD plaques and the adjacent glomeruli was performed on a Leica SP8 multiphoton microscope using a 63X glycerol immersion objective.
Slices were fixed in 4% formaldehyde in 1x phosphate buffer saline (PBS) at room temperature (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 7, 2021. ; https://doi.org/10.1101/2021.12.06.471478 doi: bioRxiv preprint for 45 min, washed in 1XPBS, blocked in 1xPBS with 0.1% TritonX100 and 2% SEA Block (ThermoFisher Scientific) for 1 hour, and sequentially incubated in primary and secondary antibodies over 2 days. Primary antibodies were as follows: tyrosine-hydroxylase (AB152, MilliporeSigma, 1:100), GFP (ThermoFisher, 1:200). To clear tissue slices, the slices were dehydrated in methanol via increasing concentrations 50%, 75%, 100% diluted in PBS -each for 1hr -and subsequently submerged in a 50:50 benzyl benzoate/benzyl alcohol (BABB): methanol solution, followed by 100% BABB. High resolution imaging of MD plaques and the adjacent glomeruli was performed on a Leica SP8 multiphoton microscope using a 63X glycerol immersion objective.
10
Immunolabeling and Imaging of Neuronal Proteins
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Primary antibodies were: βIII-tubulin (Sigma T5076, 1:500), neurofilament (Sigma N4142, 1:150), synaptotagmin (DSHB, 8 µg/ml), MAP-2 (Sigma M3696, 1:200), PSD-95 (Abcam ab18258, 1:400). Secondary antibodies conjugated with Alexa 488 and 594 (Invitrogen A11005, A11008, A10680, A11015) were used at 1:200.
We adapted standard immunocytochemistry protocols to stain and image directly the entire gels. Samples were fixed and permeabilized for 1h at 4°C in 10% formalin with 0.1% Triton X-100, blocked overnight with 5% BSA in PBS, and washed with PBS (twice 1h, once overnight). They were then incubated in primary antibody (overnight at RT with gentle shaking, dilution with 3% BSA in PBS), washed with PBS, incubated overnight in secondary antibody, washed with PBS, stained with DAPI 0.3 µM in PBS for 1h, and finally washed with PBS.
Imaging of immunostained samples was performed on a Leica SP8 multiphoton microscope with a 20x/0.95 NA water objective, typically exciting at 710 nm and 1100 nm using MaiTai Deepsee and Insight Deepsee fs-lasers respectively (Spectra Physics). The resulting stacks were edited in Fiji to apply a MIP, and adjust the color display (brightness/contrast/gamma). When salt and pepper noise was present, median filtering over < 2 px was applied.
We adapted standard immunocytochemistry protocols to stain and image directly the entire gels. Samples were fixed and permeabilized for 1h at 4°C in 10% formalin with 0.1% Triton X-100, blocked overnight with 5% BSA in PBS, and washed with PBS (twice 1h, once overnight). They were then incubated in primary antibody (overnight at RT with gentle shaking, dilution with 3% BSA in PBS), washed with PBS, incubated overnight in secondary antibody, washed with PBS, stained with DAPI 0.3 µM in PBS for 1h, and finally washed with PBS.
Imaging of immunostained samples was performed on a Leica SP8 multiphoton microscope with a 20x/0.95 NA water objective, typically exciting at 710 nm and 1100 nm using MaiTai Deepsee and Insight Deepsee fs-lasers respectively (Spectra Physics). The resulting stacks were edited in Fiji to apply a MIP, and adjust the color display (brightness/contrast/gamma). When salt and pepper noise was present, median filtering over < 2 px was applied.
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