Mice were perfused transcardially with cold PBS, followed by 4% paraformaldehyde (PFA) in PBS. Spinal cords were extracted and kept in 4% PFA at 4°C overnight and then left in sucrose (30%). Slices were collected at 20/40 micrometer of thickness in the cryostat. For immunostaining, each slice was placed in PBST (PBS + 0.2% Triton X-100) with 10% serum for 2 h at room temperature and then incubated with primary antibody at 4°C over night (mouse anti-GABA 1∶500, Abcam; chicken/rabbit anti-GFP 1∶500, Invitrogen; rat anti-glycine 1∶1000, ImmunoSolution). Slices then underwent three wash steps for 10 min each in PBST, followed by 2 h incubation with secondary antibody (1∶200 AlexaFlour 488 anti-chicken/rabbit, Invitrogen; 1∶200 AlexaFlour 546 anti-rat, Invitrogen; 1∶200 AlexaFlour 647 anti-mouse, Invitrogen). Slices were then incubated and mounted with DAPI (vectashield) on microscope slides and imaged using a confocal microscope (Zeiss LSM 5 Pascal Exciter). For cell counting, we analyzed (20× magnification) the dorsal, middle, and ventral regions of the lower thoracic spinal cord of 4 animals. ChR2-EYFP+/GLY+/GAD+ neurons were identified by means of co-localization of respectively the membrane/soma with DAPI.
Lsm 5 pascal exciter
Manufactured by Zeiss
Sourced in Germany
The LSM 5 Pascal Exciter is a laser scanning microscope component manufactured by Zeiss. It serves as an excitation source, providing the necessary light to stimulate samples during microscopic analysis. The core function of this product is to generate the required wavelengths of light to enable the imaging and examination of specimens in a variety of research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield hardset mounting media, by Vector Laboratories (3 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Ab41172, by Abcam (2 mentions)
Ab23832, by Abcam (2 mentions)
Ab55980, by Abcam (2 mentions)
Ab5164, by Merck Group (2 mentions)
Ab5166, by Abcam (2 mentions)
Amaxa nucleofector, by Lonza (1 mentions)
Image browser software, by Zeiss (1 mentions)
Mab377, by Merck Group (1 mentions)
G9269, by Merck Group (1 mentions)
Alexa fluor 405 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
7 protocols using lsm 5 pascal exciter
1
Spinal Cord Immunostaining for Inhibitory Neuron Markers
2
Immunohistochemical Evaluation of Cholinergic Receptors in SOM-TD Mice
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SOM-TD mice (P34, Supplementary Fig. 6f ) were anesthetized with 4% isoflurane and perfused transcardially with saline followed by chilled 4 % paraformaldehyde in 0.1 M PBS. The brains were then postfixed in 4 % paraformaldehyde in 0.1 M PBS (<4°C) overnight. The fixed brains were sectioned into 50 μm visual cortical slices with a vibratome and then blocked in 10% normal goat serum with 1% triton in PBS (1 hour, room temperature) before being stained with rabbit anti-M1 and anti-M2 (1:200, Millipore, AB5164, AB5166) or rabbit anti-nAChR alpha4 and rabbit anti-nAChR beta2 or rabbit anti-nAChR alpha7 (1:200, Abcam, ab41172, ab55980, ab23832)67 (link) overnight (< 4 °C). This was followed by a 3 hour incubation in Alexa Fluor 488 goat anti-rabbit (1:200, Invitrogen, A11034) before being mounted on a glass slide with the Vectashield Hardset mounting media (Vector Labs). The slides were imaged using a confocal microscope (Zeiss LSM 5 Pascal Exciter) and the images were analyzed for co-localization of tdTomato positive SOM neurons and the respective cholinergic receptors stains.
3
Tracking Astrocyte Focal Adhesion Dynamics
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Primary astrocytes were transfected with RFP-CrkI (a gift from N. Mochizuki, National Cerebral and Cardiovascular Center, Osaka, Japan; Nagashima et al., 2002 (link)) on day 11 using Amaxa nucleofector (Lonza) and replated the next day into an 8-well, glass-bottom chamber (Iwaki) that was coated by 5 µg/ml laminin; 2 d later, and 2 h after a wound was made in the astrocyte monolayer using a P10 pipette tip, the fluorescent cells at the edge of the wound were recorded using a confocal microscope (LSM5, Pascal Exciter; ZEISS) that was equipped with a heat- (37°C) and gas-controlled incubation chamber (5% CO2; Tokai Hit), which was coupled to a heated-motor stage. The objective lens (EC Plan-Neofluar, 10×, NA 0.3) was maintained at 37°C and was used to acquire a Z-stack, time-lapse series with the following parameters: zoom 4× every 15 min, seven Z-stacks (every 5 µm). Z-projections were performed with the Image Browser software (ZEISS). The formation of FA at the leading edge was tracked until disappearance using the Manual Tracking plugin of the ImageJ software (1.48). The data were collected in six different recording sessions using three independent, primary cultures of astrocytes.
4
Evaluating Resin-Dentin Interface Using CLSM
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After completion of the restorative procedures, two longitudinal sections were made along the long axis of the teeth in the middle portion of the tooth to obtain a tooth section measuring approximately 0.5 mm thickness using a slow speed diamond disc under copious water supply (
Fig. 3 ). The sectioned samples were further grinded on a carborundum stone to achieve a smooth uniform, even surface. The samples were pat dried with a paper towel and mounted on the glass slide with a coverslip.
The resin-dentin interface of the tooth samples in both test and control groups was examined to observe resin tag length and hybrid layer thickness by using CLSM (LSM 5 Pascal Exciter, Zeiss, Germany) under ×10 magnification and Argon laser illumination at 50% intensity with 514 nm excitation wavelength. Confocal slits were set at 25 μm with a 536 nm long-pass filter (
Fig. 4 ).
The images were analyzed and the average of three readings was recorded for each parameter. The resin tag length and hybrid layer thickness were measured by using Image Browser Software (Zeiss, Germany) in micrometer by a single trained examiner. Prior to the experimental study, intraexaminer calibration was done and the reliability was verified at two different time intervals in two weeks gap. Intraclass correlation coefficient (ICC) reliability index value was estimated to be greater than 0.9.
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After completion of the restorative procedures, two longitudinal sections were made along the long axis of the teeth in the middle portion of the tooth to obtain a tooth section measuring approximately 0.5 mm thickness using a slow speed diamond disc under copious water supply (
The resin-dentin interface of the tooth samples in both test and control groups was examined to observe resin tag length and hybrid layer thickness by using CLSM (LSM 5 Pascal Exciter, Zeiss, Germany) under ×10 magnification and Argon laser illumination at 50% intensity with 514 nm excitation wavelength. Confocal slits were set at 25 μm with a 536 nm long-pass filter (
The images were analyzed and the average of three readings was recorded for each parameter. The resin tag length and hybrid layer thickness were measured by using Image Browser Software (Zeiss, Germany) in micrometer by a single trained examiner. Prior to the experimental study, intraexaminer calibration was done and the reliability was verified at two different time intervals in two weeks gap. Intraclass correlation coefficient (ICC) reliability index value was estimated to be greater than 0.9.
5
Immunohistochemical Evaluation of Cholinergic Receptors in SOM-TD Mice
6
Immunohistochemical Localization of Na,K-ATPase
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Skeletal muscles from control and HS rats fixed in 4% formaldehyde were washed with the phosphate-buffered salt solution (PBS) containing (in mmol/L): NaCl 137, KCl 2.7, Na2HPO4 8.2, KH2PO4 1.8, at pH 7.4. Then, unreacted fixative was quenched with 25 mmol/L glycine in PBS for 15 minutes. Muscle fibers were washed 3 times with PBS, permeabilized with 0.1% Triton-X in PBS for 1 h and incubated with primary α2 isoform of the Na,K-pump antibody (1 : 100, Millipore, USA) overnight at 5°C. After 3 wash, muscles were incubated in the dark with Alexa-488 fluorescent conjugated secondary antibody for 1 h at room temperature (1 : 2000; Invitrogen, USA). After washing the preparation was transferred to the confocal microscope (LSM-5 Pascal Exciter, Zeiss, Germany). The emission signal at 530 nm (after excitation at 488 nm) was stored on the computer for later analyses of fluorescence intensity using ImageJ (NIH, USA).
7
AAV-mediated GFAP-ChR2 expression in mouse brain
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C57/BL6 mice transfected with AAV2.5-GFAP-ChR2–mCherry were overdosed with pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde (n=3 mice). The brain was extracted and kept in paraformaldehyde overnight. The brain was sectioned in 50 μm slices that were then blocked in 10% normal goat serum with 1% triton in phosphate-buffered saline (1 h, room temperature) and stained for rabbit anti-GFAP (1:200, Sigma, G9269) and mouse anti-NeuN (1:250, Millipore, MAB377) overnight (<4 °C). This was followed by a 3-h incubation in Alexa Fluor 488 goat anti-rabbit (1:200, Invitrogen, A11034) and Alexa Fluor 405 goat anti-mouse (1:200, Invitrogen, A31553) before being mounted on a glass slide with the Vectashield Hardset mounting media (Vector Labs). The slides were imaged using a confocal microscope (Zeiss LSM 5 Pascal Exciter).
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