Femurs were longitudinally cut in half, then stained, and deep imaged as described previously21 (link). The staining solution contained 10% DMSO, 0.5% IgePal630 (Sigma), and 5% donkey serum (Jackson Immuno) in PBS. Half bones were stained for 3 days at room temperature with primary antibodies. Then specimens were washed 3 times in PBS at room temperature for one day and put into staining solution containing secondary antibodies for 3 days followed by a one-day wash. Antibodies used for whole mount staining included chicken anti-GFP (Aves Labs, 1:200), anti-tdTomato (Takara, 1:200), Alexa Fluor 647-AffiniPure F(ab′)2 Fragment Donkey Anti-Chicken IgY (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (1:250), Alexa Fluor 488-AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (all from Jackson ImmunoResearch, 1:250), and 555--conjugated donkey anti-goat antibody (Invitrogen, 1:250). Images were acquired using a Leica SP8 confocal microscope. Images were rendered in 3D and analyzed using Bitplane Imaris v7.7.1.
Anti tdtomato
Manufactured by Takara Bio
The Anti-tdTomato is a laboratory reagent used for the detection and analysis of tdTomato, a red fluorescent protein commonly used as a reporter in biological research. This antibody specifically binds to and labels tdTomato, enabling its identification and quantification in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Donkey serum, by Jackson ImmunoResearch (2 mentions)
Igepal630, by Merck Group (2 mentions)
Alexa fluor 647 affinipure f ab 2 fragment donkey anti chicken igy, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 affinipure f ab 2 fragment donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
555 conjugated donkey anti goat antibody, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Chicken anti gfp, by Aves Labs (2 mentions)
Anti vglut2, by Synaptic Systems (1 mentions)
Anti map2, by Merck Group (1 mentions)
Anti c fos, by Santa Cruz Biotechnology (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
B6 129s6 gt rosa 26sortm9 cag tdtomato hze j, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
B6.129 leprtm2 cre rck j, by Jackson ImmunoResearch (1 mentions)
Anti ck5, by Abcam (1 mentions)
4 protocols using anti tdtomato
1
Whole-mount Imaging of Bone Samples
2
Whole-mount Imaging of Bone Samples
3
Immunohistochemical analysis of mouse hippocampus
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Mice were terminally anesthetized, transcardially perfused with saline and 20 ml of 4% paraformaldehyde (PFA), and brains were post-fixed overnight. Coronal sections (100 μm) of the hippocampus were collected and permeabilized in 0.4% Triton in PBS (PBST) for 30 min. Sections were then blocked for 30 min with 5% horse serum in PBST and incubated overnight (4°C) with primary antibody in 5% horse serum/PBST. After extensive washing, sections were incubated with the appropriate secondary antibody conjugated with Alexa 488, 568 or 647 (Molecular Probes), for 2hrs at room temperature. They were then washed in PBST (2 × 10 min) and mounted with Dapi Fluoromount-G (SouthernBiotech). The primary antibodies used were: anti-c- fos (1:500, Santa-Cruz), anti-tdTomato (1:500, Clontech), anti-MAP2 (1:500, Sigma), anti-ABBA/Mtss1L (1:500, Millipore), anti-VGLUT2 (1:500, Synaptic Systems) and anti-beta-galactosidase (1:20, Developmental Studies Hybridoma Bank). All antibodies have been well characterized in prior studies in our laboratory, and staining was not observed when the primary antibody was omitted.
4
Visualizing LepR-expressing cells in the mammary gland
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We used the LepR-reporter mouse to visualize LepR-expressing cells. LepR-reporter mice were generated by breeding the LepRb-IRES-Cre mouse (B6.129-Leprtm2(cre)Rck/J, Jackson Laboratories) with the Cre-inducible tdTomato-reporter mouse (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, Jackson Laboratories). This mouse model has been validated6 (link)45 (link). Only cells that express the long form of LepR contain the red fluorescent tdTomato protein in the LepR-reporter mouse. To identify the localization of LepR-expressing cells in the mammary tissue, the inguinal glands of four pregnant LepR-reporter mice were dissected at specific developmental time-points (16–18 days of pregnancy). The tissue was fixed in 4% paraformaldehyde overnight at 4 °C and processed for hematoxylin-eosin staining or immunostaining according to the following procedures: 4-μm sections were deparaffinized, rehydrated, and subjected to 10 mM citrate buffer pH 6,0 antigen retrieval for 30 min at 95 °C. Anti-tdTomato (Clontech) or Anti-CK5 (Abcam) primary antibodies were incubated overnight at 4 °C at 1:200 dilution, and Alexa fluor 488-conjugated and Alexa fluor 633-conjugated secondary antibodies were used, respectively, at 1:750 dilutions. Samples were mounted in Prolong Gold antifade reagent with DAPI (Molecular Probes).
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