a1rsi inverted confocal microscope, by Nikon - Product details

1

Confocal Imaging of Arabidopsis Embryo Sac and Pollen

Check if the same lab product or an alternative is used in the 5 most similar protocols

Analysis of embryo sac development was conducted using confocal microscopy based on [59 (link)]. Pistils were dissected from FG2, FG3, FG4 and mature stages of flowers from Col-0, nerd1-2 and nerd1-4 and fixed in 4% glutaraldehyde and 12.5mM cocadylate, PH = 6.9 for two hours at room temperature. Pistils were dehydrated in 20%, 40%, 60%, 80% and 100% ethanol for 10 min each. Samples were then cleared in a 2:1 mixture of benzyl benzonate: benzyl alcohol for 2–4 hours and mounted in immersion oil for imaging. Images were captures using a Nikon A1Rsi inverted confocal microscope under 60x oil objectives and excitation with a 561nm laser.
Pollen was released from tetrad, microspore, bi-cellular, tri-cellular and mature stage anthers from Col-0, nerd1-2 and nerd1-4. Samples were incubated in 1ug/ml DAPI (4′,6-diamidino-2-phenylindole) staining solution for 2hrs and imaged using a Nikon A1Rsi inverted confocal microscope under 60x oil objectives with DAPI excited by a 405nm laser.

Yuan J, & Kessler S.A. (2019). A genome-wide association study reveals a novel regulator of ovule number and fertility in Arabidopsis thaliana. PLoS Genetics, 15(2), e1007934.

+ Open protocol

+ Expand

2

Tissue Fixation and Immunostaining Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

All tissues were fixed in 4% paraformaldehyde for either one hour at room temperature for frozen processing or overnight at 4°C for paraffin processing. For frozen sections, tissue was protected in 30% sucrose overnight at 4°C, then embedded in OCT (Tissue-Tek), and cut at 10 μm. For paraffin sections, tissue was processed through a graded ethanol series, followed by xylene, and then embedded in paraffin and cut at 7 μm. Tissue culture cells were fixed for 15 minutes at room temperature and stained directly. For staining, frozen slides were thawed to room temperature and rehydrated in PBS, while paraffin slides were deparaffinized and subjected to antigen retrieval. Slides were blocked in 5% normal donkey serum (Jackson Immuno Research) in PBS plus 0.5% Triton-X for 30 minutes at room temperature. Primary antibodies (listed in Methods Table 1) were diluted in blocking buffer and incubated overnight at 4°C. Slides were washed in PBS and incubated with secondary antibody for one hour at room temperature, and coverslips were mounted using Fluoromount-G (Southern Biotech). Confocal images were captured on a Nikon A1Rsi inverted confocal microscope.

McCracken K.W., Catá E.M., Crawford C.M., Sinagoga K.L., Schumacher M., Rockich B.E., Tsai Y.H., Mayhew C.N., Spence J.R., Zavros Y, & Wells J.M. (2014). Modeling human development and disease in pluripotent stem cell-derived gastric organoids. Nature, 516(7531), 400-404.

+ Open protocol

+ Expand

3

Whole-mount confocal imaging protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Confocal imaging was performed on Nikon A1Rsi inverted confocal microscope. For wholemount imaging, embryos were dehydrated in methanol and cleared in Murray’s clear (2:1 benzyl benzoate: benzyl alcohol) just prior to imaging. After staining, slides were mounted with Fluoromount G (SouthernBiotech), and air-dried overnight at room temperature.

McCracken K.W., Zhang X, & Wells J.M. (2017). Wnt/β-catenin promotes gastric fundus specification in mice and humans. Nature, 541(7636), 182-187.

+ Open protocol

+ Expand

4

Neural Crest Cell Migration Dynamics

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dorsal neural tubes were harvested from HH8⁺- to HH9-control and -ta² embryos, and cultured in neural crest cell medium (Bajpai et al., 2010 (link)) in an 8-well chamber slide (μ–slide 8 well, 80821, Ibidi, Verona, WI, USA) that had been coated with fibronectin. The remaining portion of the embryo was used for genotyping. Explants were incubated at 37°C for 5-6 h. Approximately 30 min before imaging, neural crest cell medium was replaced with 3 μM Di-8-Anneps (a gift from Brian Sirosky, CCHMC, Cincinnati, OH, USA) in order to fluorescently label cell membranes. Explants were imaged for 12 h at 2-min intervals using a Nikon A1Rsi inverted confocal microscope. Videos were analyzed using Imaris software.

Schock E.N., Chang C.F., Struve J.N., Chang Y.T., Chang J., Delany M.E, & Brugmann S.A. (2015). Using the avian mutant talpid2 as a disease model for understanding the oral-facial phenotypes of oral-facial-digital syndrome. Disease Models & Mechanisms, 8(8), 855-866.

+ Open protocol

+ Expand

5

Whole-mount confocal imaging protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Confocal imaging was performed on Nikon A1Rsi inverted confocal microscope. For wholemount imaging, embryos were dehydrated in methanol and cleared in Murray’s clear (2:1 benzyl benzoate: benzyl alcohol) just prior to imaging. After staining, slides were mounted with Fluoromount G (SouthernBiotech), and air-dried overnight at room temperature.

McCracken K.W., Zhang X, & Wells J.M. (2017). Wnt/β-catenin promotes gastric fundus specification in mice and humans. Nature, 541(7636), 182-187.

+ Open protocol

+ Expand

6

Immunofluorescent Staining of Kidney Cultures

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recipient kidneys together with the trans-well filter were fixed in 4% PFA for 6hrs, washed with PBS and blocked in PBS-BB (PBS containing 1% BSA, 0.2% powdered skim milk and 0.3% Triton-100) overnight at 4°C. After blocking, kidney cultures were incubated with primary antibodies (see Supplemental information for antibodies and dilutions used) in the PBS-BB for 48hrs to allow sufficient penetration. Primary antibodies were washed off for 48hrs at 4°C followed by overnight secondary antibody incubation and another 48hr of wash at 4°C. Stained kidneys together with the trans-well filter were mounted with ProLong® Gold Antifade Reagent (Life Technologies) and allowed to cure overnight before imaging. Confocal imaging was performed on a Nikon A1Rsi inverted confocal microscope with fixed and stained kidney cultures on the glass slide. For each injection site, samples were imaged on a 40X water-immersion lens using a pinhole of 1.2μm on all channels at the resolution of 1024X1024. The z-stacks were taken with an interval of 1.5μm over 50-80μm to produce a stack of consecutive images with 1/3 overlaps.

Chen S., Brunskill E.W., Potter S.S., Dexheimer P.J., Salomonis N., Aronow B.J., Hong C.I., Zhang T, & Kopan R. (2015). Intrinsic age-dependent changes and cell-cell contacts regulate nephron progenitor lifespan. Developmental cell, 35(1), 49-62.

+ Open protocol

+ Expand

7

Immunofluorescence Analysis of Liver Inflammation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Liver tissues were embedded in OCT compound (Sakura Finetek). OCT compound-embedded tissues were cut into 5-μm sections and fixed in 4% paraformaldehyde. After rinsing with PBS, sections were permeabilized and treated with blocking buffer (0.2% Triton X-100, 0.2% bovine serum albumin (BSA), and 0.1% normal goat serum in PBS). After the blocking process, sections were incubated with FITC-conjugated anti-TNFα antibody (1 : 200 dilution; eBioscience), FITC-conjugated anti-IL-6 antibody (1 : 200 dilution; eBioscience), and APC-conjugated anti-F4/80 antibody (1 : 1000 dilution; eBioscience) in blocking buffer at 4 °C overnight. After that, sections were washed with PBS and incubated with DAPI for 5 min. After rinsing with PBS, the sections were mounted with mounting fluid and visualized under an A1Rsi inverted Confocal Microscope (Nikon).

Ito S., Tanaka Y., Oshino R., Okado S., Hori M, & Isobe K.I. (2016). GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation. Cell Death & Disease, 7(5), e2219-.

+ Open protocol

+ Expand

8

Hepatoblasts Proliferation Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Hepatoblasts proliferation was measured by using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime® Biotechnology, China, C0071S). According to the manufacturer’s recommendations, cells were treated with medium containing 10 μM EdU for 3 h to label proliferating cells. After incubation, cells were washed three times with Knockout DMEM basic medium and fixed in 4% PFA before permeabilization with PBS containing 0.5% Triton X-100 for 15 min. Next, the cells were treated with Click Reaction Buffer and were incubated at room temperature in the dark for 30 min. After washing, cell nuclei were stained with Hoechst 33342 (1:2000) for 20 min. Stained samples were observed under a Nikon A1Rsi inverted confocal microscope.

Zhang Y., Guo A., Lyu C., Bi R., Wu Z., Li W., Zhao P., Niu Y., Na J., Xi J.J, & Du Y. (2021). Synthetic liver fibrotic niche extracts achieve in vitro hepatoblasts phenotype enhancement and expansion. iScience, 24(11), 103303.

+ Open protocol

+ Expand

9

Tissue Fixation and Immunostaining Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

All tissues were fixed in 4% paraformaldehyde for either one hour at room temperature for frozen processing or overnight at 4°C for paraffin processing. For frozen sections, tissue was protected in 30% sucrose overnight at 4°C, then embedded in OCT (Tissue-Tek), and cut at 10 μm. For paraffin sections, tissue was processed through a graded ethanol series, followed by xylene, and then embedded in paraffin and cut at 7 μm. Tissue culture cells were fixed for 15 minutes at room temperature and stained directly. For staining, frozen slides were thawed to room temperature and rehydrated in PBS, while paraffin slides were deparaffinized and subjected to antigen retrieval. Slides were blocked in 5% normal donkey serum (Jackson Immuno Research) in PBS plus 0.5% Triton-X for 30 minutes at room temperature. Primary antibodies (listed in Methods Table 1) were diluted in blocking buffer and incubated overnight at 4°C. Slides were washed in PBS and incubated with secondary antibody for one hour at room temperature, and coverslips were mounted using Fluoromount-G (Southern Biotech). Confocal images were captured on a Nikon A1Rsi inverted confocal microscope.

McCracken K.W., Catá E.M., Crawford C.M., Sinagoga K.L., Schumacher M., Rockich B.E., Tsai Y.H., Mayhew C.N., Spence J.R., Zavros Y, & Wells J.M. (2014). Modeling human development and disease in pluripotent stem cell-derived gastric organoids. Nature, 516(7531), 400-404.

+ Open protocol

+ Expand

10

Confocal Microscopy Imaging Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Samples were mounted in VECTASHIELD Antifade Mounting Medium. Images were obtained on a Nikon A1R-Si inverted confocal microscope with 4 line laser unit (405/488/561/640) and with a 60X oil objective. Z stacks were acquired with a step size of 0.125 μm between optical sections.

Ji W., Wu L.F., & Altschuler S.J. (2020). On the role of photoreceptor identity in controlling accurate wiring of theDrosophilavisual circuit.

+ Open protocol

+ Expand

A1rsi inverted confocal microscope

Lab products found in correlation

14 protocols using a1rsi inverted confocal microscope

Confocal Imaging of Arabidopsis Embryo Sac and Pollen

Tissue Fixation and Immunostaining Protocol

Whole-mount confocal imaging protocol

Neural Crest Cell Migration Dynamics

Whole-mount confocal imaging protocol

Immunofluorescent Staining of Kidney Cultures

Immunofluorescence Analysis of Liver Inflammation

Hepatoblasts Proliferation Quantification

Tissue Fixation and Immunostaining Protocol

Confocal Microscopy Imaging Protocol

About PubCompare

Ready to get started?