Inverted confocal microscope

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An inverted confocal microscope is a type of optical microscope that uses a focused laser beam to scan a sample. It allows for high-resolution imaging of specimens by rejecting out-of-focus light, resulting in a sharper, more detailed image. The core function of an inverted confocal microscope is to provide a detailed, three-dimensional view of a sample.

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Lab products found in correlation

Fluorescent conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Rabbit anti active caspase 3, by Cell Signaling Technology (1 mentions) Rabbit anti ph3, by Merck Group (1 mentions) Rabbit anti gm130, by Abcam (1 mentions) Mouse anti cytochrome c, by BD (1 mentions)

3 protocols using inverted confocal microscope

Intestine Immunostaining Protocol

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For standard immunostainings, intestines were dissected in 1 × PBS (10 mM NaH₂PO₄/Na₂HPO₄, 175 mM NaCl, pH 7.4), and fixed in 4% paraformaldehyde for 25 min at room temperature. Samples were washed with 1 x PBT (0.1% Triton X‐100 in 1 × PBS) and blocked in 3% BSA in 1 × PBT for 45 min. Primary antibodies were added to the samples and incubated at 4°C overnight. The following primary antibodies were used: mouse mAb anti‐Dl (C594.9B, 1:50, developed by S. Artavanis‐Tsakonas, DSHB), mouse mAb anti‐Prospero (MR1A, 1:100, developed by Chris Doe, DSHB), rabbit anti‐pH 3 (1:2000, Millipore), rabbit anti‐active caspase 3 (1:1000, Cell Signalling), and rabbit anti‐Yun (1:1000).²⁵ Primary antibodies were detected by fluorescent‐conjugated secondary antibodies from Jackson ImmunoResearch Laboratories. Secondary antibodies were incubated for 2 h at room temperature. DAPI (Sigma‐Aldrich; 0.1 μg/ml) was added after secondary antibody staining. The samples were mounted in mounting medium (70% glycerol containing 2.5% DABCO). All images were captured using a Zeiss inverted confocal microscope and were processed in Adobe Photoshop and Illustrator.

Ren X., Zhao H., Shi L., Li Z., Kong R., Ma R., Jia L., Lu S., Wang J., Dong M., Wang Y, & Li Z. (2022). Phosphorylation of Yun is required for stem cell proliferation and tumorigenesis. Cell Proliferation, 55(5), e13230.

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Immunostaining of Intestinal Tissues

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For standard immunostainings, intestines were dissected in 1 × PBS (10 mM NaH₂PO₄/Na₂HPO₄, 175 mM NaCl, pH7.4), and fixed in 4% paraformaldehyde for 25 min at room temperature. Samples were washed with 1 × PBT (0.1% Triton X-100 in 1 × PBS) and blocked in 3% BSA in 1 × PBT for 45 min. Primary antibodies were added to the samples and incubated at 4 °C overnight. The following primary antibodies were used: mouse mAb anti-Dl (C594.9B, 1:50, developed by S. Artavanis-Tsakonas, DSHB), mouse mAb anti-Prospero (MR1A, 1:100, developed by Chris Doe, DSHB), mouse anti-Cytochrome C (1:25, BD Biosciences), rabbit anti-pH3 (pSer10, Millipore, 1:2000), rabbit anti-GM130 (Abcam, 1:200), rabbit anti-Cova (this study, 1:100). Primary antibodies were detected using fluorescent-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories. Secondary antibodies were incubated for 2 h at room temperature. DAPI (Sigma-Aldrich; 0.1 μg/ml) was added after secondary antibody stainings. The samples were mounted in mounting medium (70% glycerol containing 2.5% DABCO). All images were captured using a Zeiss inverted confocal microscope and were processed in Adobe Photo-shop and Illustrator.

Ma M., Zhao H., Zhao H., Binari R., Perrimon N, & Li Z. (2016). Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila. Developmental biology, 411(2), 207-216.

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Immunofluorescence Analysis of Mutant NLGN3 Proteins

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PC12 Tet-On cells expressing either WT or mutant NLGN3 proteins were plated on glass coverslips coated with 0.1 μg/ml poly-L-lysine and grown in DMEM. Cells were fixed in 4% (w/v) paraformaldehyde in PBS, washed and incubated with blocking buffer (PBS, 2% normal goat serum, 0.5% BSA and 50 mM glycine) [4 (link)]. Primary antibody incubation was performed with either rabbit anti-FLAG or anti-FLAG M2 mouse monoclonal antibody diluted 1:500 in combination with the anti-calnexin or anti-CHOP diluted 1:200 in 5× diluted blocking buffer. Secondary antibodies were diluted 1:500 in the same buffer. Nuclei were stained with DAPI.
Four or five images for each sample were captured at room temperature using a Nikon inverted confocal microscope with ×40 and ×63 objective lenses and processed using ImageJ and Adobe Photoshop software.

Ulbrich L., Favaloro F.L., Trobiani L., Marchetti V., Patel V., Pascucci T., Comoletti D., Marciniak S.J, & De Jaco A. (2016). Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system. Biochemical Journal, 473(Pt 4), 423-434.

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