We performed whole-mount immunostaining followed by confocal microscopy to examine the infiltration of F4/80+ macrophages in adipose tissue. The mice were perfused with PBS before the collection of inguinal white adipose tissue. The tissue depots were subdivided into 0.5–0.75 cm3 sized pieces, fixed in 1% paraformaldehyde (PFA) for 30 min at room temperature with gentle rocking, and washed in PBS three times for 10 min under gentle rocking. The tissue was blocked for 30 min in 5% BSA in PBS, then incubated with primary anti-F4/80 antibodies (Thermo Fisher Scientific, A3-1, MA1-91124) diluted 1:100 in blocking buffer overnight at 4 °C. The samples were washed with times with PBS before incubation with secondary anti-rat-Alexa 488 antibodies (Thermo Fisher Scientific, A-21208) diluted 1:350 in blocking buffer for 1 h at room temperature with gentle rocking. After three washes with PBS, the samples were incubated for 20 min in a lipid- and nucleus-staining cocktail composed of 1 µg ml−1 DAPI (Sigma-Aldrich, D9542) and 0.25 µg ml−1 BODIPY (Thermo Fisher Scientific, D3922) and washed three times in PBS before imaging. The images were taken on an inverted scanning confocal microscope (Zeiss LSM 700 Inverted) and assembly of 3D reconstructions was accomplished by taking z-stack images.
Lsm700 inverted
Manufactured by Zeiss
Sourced in Germany
The LSM700 inverted is a laser scanning microscope designed for imaging and analysis of biological samples. It features an inverted optical configuration, allowing for easy access to the sample and integration with a wide range of live cell imaging accessories. The core function of the LSM700 inverted is to provide high-resolution, confocal imaging capabilities for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Zen lite 2011 microscope software, by Zeiss (2 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Acidic tyrode s solution, by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Pfa 4, by Merck Group (1 mentions)
Rabbit anti ha antibody, by Merck Group (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit cy5, by Jackson ImmunoResearch (1 mentions)
Plan apochromat 63x 1.4 oil objective, by Zeiss (1 mentions)
Clear bottom 96 well plate, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Er tracker red dye, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using lsm700 inverted
1
Macrophage Infiltration in Adipose Tissue
2
Quantifying Neuronal Cell Death Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell death was also quantified by the
total count of cells. The remaining treated neurons in the 96-well
plate were fixed at the indicated times in 4% PFA (para-formaldehyde)
for 20 min at RT and then washed away using 1× PBS. Neurons were
permeabilized and blocked in a 1× PBS (pH 7.4) solution composed
of 0.1% Triton X-100 and 3% BSA for 30 min at RT. The cells were then
stained using an antibody against the nuclear neuronal marker protein
NeuN (1:2000) and an antibody against the microtubule-associated protein
MAP2 (1:2000). The nucleus was counterstained with DAPI at 1:1000
(Sigma-Aldrich, Switzerland). Cells were washed 2 times in 1×
PBS afterward, and the plate was then examined with a microscope (LSM
700 inverted, Zeiss) with a 20× objective and analyzed using
ImageJ (U.S. NIH, Bethesda, MD, USA; RRID, SCR_003070).
Results
were plotted using Origin 2021. Unpaired t test and
ANOVA test were used for the statistical analysis.
total count of cells. The remaining treated neurons in the 96-well
plate were fixed at the indicated times in 4% PFA (para-formaldehyde)
for 20 min at RT and then washed away using 1× PBS. Neurons were
permeabilized and blocked in a 1× PBS (pH 7.4) solution composed
of 0.1% Triton X-100 and 3% BSA for 30 min at RT. The cells were then
stained using an antibody against the nuclear neuronal marker protein
NeuN (1:2000) and an antibody against the microtubule-associated protein
MAP2 (1:2000). The nucleus was counterstained with DAPI at 1:1000
(Sigma-Aldrich, Switzerland). Cells were washed 2 times in 1×
PBS afterward, and the plate was then examined with a microscope (LSM
700 inverted, Zeiss) with a 20× objective and analyzed using
ImageJ (U.S. NIH, Bethesda, MD, USA; RRID, SCR_003070).
Results
were plotted using Origin 2021. Unpaired t test and
ANOVA test were used for the statistical analysis.
3
Immunofluorescent Analysis of Oocyte DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oocytes obtained by collagenase-EDTA digestion were fixed in 4% paraformaldehyde (PFA). After the fixed oocytes were permeabilized with 0.5% Triton X-100 at room temperature (RT) for 20 min, they were blocked with 1% BSA-supplemented PBS for 1 h and incubated at 4 °C with anti-γ-H2AX (phospho S139) antibody (1:500 dilution) and anti-p-ATM (S1981) antibody (1:500 dilution) overnight followed by an incubation with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 goat anti-mouse IgG (Invitrogen Molecular Probes, Carlsbad, CA, USA) for 1 h at RT. DNA was stained with DAPI (1 μg/ml in PBS). Finally, the oocytes were mounted on glass slides with DABCO-containing mounting medium and examined by laser-scanning confocal microscopy (Zeiss LSM700 Inverted).
4
Chromosome Spread Analysis of Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For chromosome spreads, the oocytes isolated from PD14 Cdk1+/SAF and OoCdk1+/AF; Zp3-Cre ovaries were first freed of the zona pellucida with acidic Tyrode's solution (Sigma-Aldrich). After a brief recovery in M2 medium, the oocytes were transferred onto glass slides and fixed in a solution of 1% PFA in distilled H2O (pH 9.2) containing 0.15% Triton X-100 and 3 mM dithiothreitol. The slides were left to dry and then blocked with 1% BSA in PBS for 1 h at RT. The spreads were incubated with primary antibodies against CREST and SMC3 (Abcam) overnight at 4 °C using a dilution of 1:100. After three washes, the slides were incubated with the corresponding secondary antibodies (Invitrogen) at 1:500 dilution for 1 h at RT. DNA on the slides was stained with DAPI, and the slides were mounted for observation with laser-scanning confocal microscopy (Zeiss LSM700 Inverted).
5
Identifying Spinal Cord Motor Neuron Subtypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Imaging of the stained sections was performed with a Zeiss LSM700 inverted confocal microscope, using a 40× objective. Z-stacks of 3 μm depth were taken of 8 ventral horns (VH) per animal per spinal cord level, to avoid double counting of cells.
We identified 3 subpopulations of MNs in the lumbar and cervical sections. α-MNs were distinguished from γ-MNs using the selective expression of NeuN and the presence of C boutons on the soma, [6 (link),7 (link)]. Two groups of ChAT+ α-MNs were identified—namely, α-MNs which were also NeuN-positive (ChAT+/NeuN+/C-bouton+) and αp-MNs in which NeuN is downregulated (ChAT+/NeuN−/C-bouton−). ChAT-negative MNs were identified as γ-MNs (ChAT+/NeuN−/C-bouton−) (Figure 1 ).
Image acquisition and counting were performed using Zeiss Zen 2.3 and FIJI [14 (link)]. In two mice per group, the soma area of 15 α-MNs and 10 γ-MNs was measured by defining the soma in the plane intersecting the nucleus and using the inbuilt cell size analysis in FIJI. The staining intensity of PKC-γ was normalised to the average intensity of the dorsal horn calculated in FIJI by drawing around the dorsal column. The intensity of PKC-γ in the CST was then adjusted for area and then normalised against the fluorescence of the dorsal horn [15 (link)].
We identified 3 subpopulations of MNs in the lumbar and cervical sections. α-MNs were distinguished from γ-MNs using the selective expression of NeuN and the presence of C boutons on the soma, [6 (link),7 (link)]. Two groups of ChAT+ α-MNs were identified—namely, α-MNs which were also NeuN-positive (ChAT+/NeuN+/C-bouton+) and αp-MNs in which NeuN is downregulated (ChAT+/NeuN−/C-bouton−). ChAT-negative MNs were identified as γ-MNs (ChAT+/NeuN−/C-bouton−) (
Image acquisition and counting were performed using Zeiss Zen 2.3 and FIJI [14 (link)]. In two mice per group, the soma area of 15 α-MNs and 10 γ-MNs was measured by defining the soma in the plane intersecting the nucleus and using the inbuilt cell size analysis in FIJI. The staining intensity of PKC-γ was normalised to the average intensity of the dorsal horn calculated in FIJI by drawing around the dorsal column. The intensity of PKC-γ in the CST was then adjusted for area and then normalised against the fluorescence of the dorsal horn [15 (link)].
6
Curcumin Morphological Analysis by CLSM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Morphological analysis of the curcumin was performed using a CLSM. Moreover, 30 µL of the sample was loaded on a concave glass slide and covered with a glass cover slip (0.17 mm thickness). The sample was observed by CLSM (Zeiss LSM700 inverted, Germany) and were scanned at room temperature (25 ± 1 °C) using a 20×/0.5 objective lens. Auto-fluorescence from the crystals was excited using 488 wavelength lasers for curcumin crystals.
7
Immunofluorescent Analysis of Midgut Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After dissection, individual midgut were deposited on slides, fixed in cold acetone for 15 min and rehydrated in PBS for 15 min. The midguts were then incubated for 2 h in Triton X-100 (0.2%). After washing with PBS, they were incubated for 30 min with PBS + 0.1% Tween 20 + 1% BSA. The slides were then incubated overnight at 4°C with anti-YFV-NS4B antibodies diluted 1:1000 in PBS. After washing with PBS, they were incubated for 1 h with secondary antibodies and washed with PBS. The actin network was visualized with phalloidin Alexafluor 488 (Invitrogen). After washing, nuclei were stained using Prolong gold antifade containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). All preparations were observed with a confocal microscope (ZEISS LSM 700 inverted) and images were acquired with the ZEN software.
8
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with PFA 4% (Sigma) during 20 min. Cells were permeabilized with PBS Triton X-100 (0.5%) for 15 min at RT. After washing with PBS, they were incubated for 30 min with PBS + 0.05% Tween 20 + 5% BSA. The slides were then incubated overnight at 4 °C with primary antibodies diluted in PBS. After washing with PBS, they were incubated for 1 h with secondary antibodies and washed with PBS. Nuclei were stained using PBS/NucBlue (Life Technologies, R37606). The mounting medium used is the Prolong gold (Life Technologies, P36930). All preparations were observed with a confocal microscope (ZEISS LSM 700 inverted) and images were acquired with the ZEN software.
9
Immunofluorescence Staining of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MODE-K cells were plated on collagen-coated coverslips 1 day prior to transfection. On day 2, cells were transfected using Lipofectamine (Invitrogen™, Life Technologies) according to the standard procedures and left to expand in a 37°C 5% CO2 incubator for 24 h. On the day of staining, cells were incubated with 10% normal horse serum (NHS), and thereafter with a rabbit anti-HA antibody (Sigma-Aldrich) and a goat anti-rabbit Cy5 (Jackson ImmunoResearch). Cells were blocked with NHS, stained with PE-conjugated anti-FLAG (Prozyme), fixed in 4% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were viewed using confocal microscopy (Zeiss LSM700 Inverted) and analyzed with ZEN lite 2011 microscope software (Carl Zeiss).
10
Quantifying Macrophage Fluorescence Intensity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos were imaged with Plan-Apochromat 63X/1.4 Oil Objective on a Zeiss LSM700 inverted. 10 µm stacks (0.5 µm intervals) were taken for properly staged and oriented embryos, starting 10 µm deep in the tissue. These images were converted into Z-stacks in Fiji. ROIs were drawn around macrophages (signal), copied to tissue close by without macrophages (background) and the average intensity in the green channel of each ROI was measured. For each pair of ROIs background was subtracted from signal individually. The average signal from control ROIs from one imaging day and staining was calculated and all data points from control, mutant and rescue from the same set was divided by this value. This way we introduced an artificial value called Arbitrary Unit (AU) that makes it possible to compare all the data with each other, even if they come from different imaging days when the imaging laser may have a different strength or from different sets of staining. Analysis was done on anonymized samples.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!