Fgf20β-Gal/+ embryos were pre-fixed for 2 hr in 2% PFA, 0.2% glutaraldehyde (Sigma-Aldrich, St. Louis, MO) in PBS, 4°C and then rinsed with PBS and washed 3 × 15 min with PBS, 2 mM MgCl2 (Merck, Darmstadt, Germany), 0.02% NP-40 (Sigma-Aldrich), 4°C. Subsequently, the samples were stained for 10 hr at RT, in dark with 1 mg/ml X-Gal (Thermo Fischer Scientific, Vilnius, Lithuania), 5 mM K3Fe(CN)6 (Merck, Darmstadt, Germany), 5 mM K4Fe(CN)6 (Merck, Darmstadt, Germany), 2 mM MgCl2, 0.1 % NP-40 (Calbiochem, San Diego, CA), 0.2% Na-deoxycholate (Sigma-Aldrich, St. Louis, MO) in PBS. The embryos were washed 3 × 10 min with PBS and fixed with 4% PFA in PBS at RT. After imaging, the samples were processed for paraffin blocks using standard protocols and sectioned into 5 µm sagittal sections. The sections were deparaffinized and counter stained with Nuclear Fast Red (Sigma-Aldrich, Steinheim, Germany) for 5 min, dehydrated and mounted. The sections were imaged using Axio Imager M.2 wide field microscope equipped with Plan-Neofluar 10X/0.3 objective and AxioCam HRc camera (Zeiss).
Axio imager m2 widefield microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Imager M2 is a widefield microscope designed for high-resolution imaging. It features a motorized stand, a high-intensity illumination system, and objectives with a wide range of magnification options. The microscope is capable of bright-field, phase-contrast, and fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam hrc camera, by Zeiss (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
X gal, by Thermo Fisher Scientific (1 mentions)
Np 40, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
K3fe cn 6, by Merck Group (1 mentions)
Nuclear fast red, by Merck Group (1 mentions)
K4fe cn 6, by Merck Group (1 mentions)
Plan neofluar 10x 0.3 objective, by Zeiss (1 mentions)
Na deoxycholate, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Tissue plus o c t, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mounting media, by Thermo Fisher Scientific (1 mentions)
6 protocols using axio imager m2 widefield microscope
1
Whole-Mount X-Gal Staining and Histological Analysis
2
Multi-Tissue Immunofluorescence for Pancreatic Analysis
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Human pancreata, mouse pancreata, and treated EndoC-βH1/2 cells were fixed in fixed in 4% paraformaldehyde (Electron Microscopy Services) in PBS on ice (6-hour fixation for pancreata, 15-minute fixation on slides for cells), and pancreata were embedded in either Tissue-Plus OCT (Thermo Fisher Scientific) or paraffin wax. The sections of human and rodent pancreas as well as fixed EndoC-βH1/2 cells were made permeable by 0.5% Triton treatment for 10 minutes. The paraffin sections were deparaffinized and rehydrated before citrate buffer–based antigen retrieval. Following blocking with 0.5% BSA in PBS for 120 minutes, the primary antibodies were applied overnight at 4°C. Species-matched antibodies conjugated with the Cy2, Cy3, or Cy5 fluorophores were used for secondary detection (1:1,000; Jackson ImmunoResearch). DAPI was used for nuclear staining (Southern Biotech). Immunofluorescence images were obtained using the Zeiss Axio Imager M2 widefield microscope with ApoTome. Quantification of protein signals and nuclear count were performed by ImageJ analysis. Primary antibodies are listed in Supplemental Table 5 .
3
Root Hair Phenotyping Protocol
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Root hair phenotypes were addressed in more detail using the stereo zoom microscope AxioZoom.V16 (Zeiss, Oberkochen, Germany) and when necessary, roots were also documented with differential interference optics (DIC) of an upright AxioImager M2 widefield microscope (Zeiss, Oberkochen, Germany). Root hair measurements were done on terminally elongated root hairs of control or flg22-treated wild type and HvMPK3 KO roots. In each case, a total of more than 100 root hairs from 8 to 9 roots were taken into account in one biological replicate. All measurements derived from 3 biological replicates (in total 25 roots and more than 400 root hairs per case). In all cases, statistical comparisons were done pairwise with Student’s t-test.
4
Immunofluorescence Staining of Trypanosoma Proteins
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Cells were fixed and treated as described8 (link). PBS-washed cells were fixed in 2% PFA for 10 min at 20 °C before being spread on poly(L -lysine)-coated slides and subsequently treated with 0.1% (v/v) Triton X-100 in Tris-buffered saline for 10 min at 20 °C. The anti-TbKIFC1 monoclonal antibody was obtained from the H3 hybridoma16 (link) and used at a 1:10 dilution. The anti-TbEndoG antibody was used at a 1:200 dilution. Primary antibodies were detected with an Alexa Fluor 488- or 594-conjugated goat anti-mouse or anti-rabbit IgG (Life Technologies). Cells were analysed with Zeiss Axioimager M2 widefield microscope. Deconvolution using the fast iterative algorithm (Zen Blue) was performed in Supplementary Fig. 4 .
5
Visualizing Cell Interactions via Fluorescence
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Prior to cell seeding, ASCs and HUVECs were incubated with the lipophilic dyes 3,3′dioctadecyloxacarbocyanine perchlorate (DIO, green) and 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine (DID, purple), respectively. Cells were incubated with each dye (1mL, 2μM per 1× 10 6 cells) at 37 °C for 30 min, as described by the manufacturer. The expression of osteopontin on the CSs was visualized by fluorescence microscopy. At each time point, the samples were washed with PBS and fixed at RT in formalin (10% v/v) for 15 min. Initially, CSs were incubated with 0.1% (v/v) Triton X100 in dPBS at RT for 5 min. Subsequently, the samples were rinsed and incubated in 5% (v/v) FBS/dPBS solution during 1h at RT. Afterwards, CSs were washed and incubated with 400 µl of mouse antihuman osteopontin antibody (1:100 in 5% FBS/dPBS, Biolegend, Taper) during 3h at RT. Then, the samples were incubated with the anti-mouse Alexa Fluor 555 conjugated dye solution (1:400 in 5% FBS/dPBS) for 1h in dark at RT. Lastly, the CSs were incubated with 400 µl of DAPI solution to nuclei staining (1:1000 in dPBS) during 5 min in the dark at RT. Homotypic CSs were used as control. Fluorescence microscopy analysis were performed in Axio Imager M2 widefield microscope (Carl Zeiss Microscopy GmbH) and using ZEN v2.3 blue edition.
6
Characterization of Vipr2-Cre Expression
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To characterize the Cre expression pattern, Vipr2-IRES2-Cre-neo mice were crossed with the Ai14 reporter line (Madisen et al., 2010) to generate Vipr2-IRES2-Cre-neo/wt; Ai14/wt animals.
The brains of these animals were cut into 50 µm sections by a freezing-sliding microtome (Leica SM 2101R). Sections with dLGN and V1 were then mounted on gelatin-coated slides and coverslipped with mounting media (Prolong Diamond Antifade Mounting Media, P36965, ThermoFisher).
To verify the injection location and GCaMP expression, the brain tissues from mice in dense labeling experiments were collected and sectioned with a similar procedure after all imaging sessions with additional steps of antibody staining to enhance the GCaMP signal before The sections were then imaged with Zeiss AxioImager M2 widefield microscope with a 10x/0.3 NA objective. Fluorescence from antibody enhanced GCaMP and mRuby3 were extracted from filter sets Semrock GFP-1828A (excitation 482/18 nm, emission 520/28 nm, dichroic cutoff 495 nm) and Zeiss # 20 (excitation 546/12 nm, emission 608/32 nm, dichroic cutoff 560 nm), respectively.
The brains of these animals were cut into 50 µm sections by a freezing-sliding microtome (Leica SM 2101R). Sections with dLGN and V1 were then mounted on gelatin-coated slides and coverslipped with mounting media (Prolong Diamond Antifade Mounting Media, P36965, ThermoFisher).
To verify the injection location and GCaMP expression, the brain tissues from mice in dense labeling experiments were collected and sectioned with a similar procedure after all imaging sessions with additional steps of antibody staining to enhance the GCaMP signal before The sections were then imaged with Zeiss AxioImager M2 widefield microscope with a 10x/0.3 NA objective. Fluorescence from antibody enhanced GCaMP and mRuby3 were extracted from filter sets Semrock GFP-1828A (excitation 482/18 nm, emission 520/28 nm, dichroic cutoff 495 nm) and Zeiss # 20 (excitation 546/12 nm, emission 608/32 nm, dichroic cutoff 560 nm), respectively.
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