Immunohistochemistry was performed on 5μm paraffin sections as follows: paraffin sections were first warmed to 56°C in a vacuum incubator (Isotemp Vacuum Oven, Fisher Scientific) then washed immediately twice in xylene, gradually re-dehydrated in ethanol (100%, 95%, 70%, water), and then processed for antigen retrieval in citrate buffer (10mM pH6.0)/microwave (1000 watt, 6 minutes). Samples were then washed with PBS, blocked with 1% BSA/5% donkey-serum (1 hour, room temperature), then incubated overnight at 4°C with primary antibodies (1:200 dilutions in 0.5% BSA), washed 3 times with PBS, incubated with appropriate fluorescent labeled secondary antibodies (1:1000 dilution in 0.5% BSA, Life Technologies Inc) as well as the nuclear marker DAPI (Biolegend), and slides were then mounted using Gelvatol (Sigma-Aldrich) solution prior to imaging using a Zeiss LSM 710 Confocal microscope (Carl Zeiss, Jena, Germany) under appropriate filter sets.
Fluorescent labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Fluorescent-labeled secondary antibodies are laboratory reagents designed to detect and visualize specific target proteins in biological samples. They are composed of an antibody molecule that binds to a primary antibody, with a fluorescent dye attached. This allows for the indirect detection and localization of the target protein through fluorescence microscopy or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (4 mentions)
Triton x 100, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Gelvatol, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
Fsx100 microscope, by Olympus (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Rat anti cd11b, by Thermo Fisher Scientific (2 mentions)
Rat anti cd45, by Thermo Fisher Scientific (2 mentions)
Rabbit anti neun, by Abcam (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Chicken anti gfap, by Abcam (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Oligomycin, by Cayman Chemical (2 mentions)
Antimycin, by Merck Group (2 mentions)
Valinomycin, by Bio-Techne (2 mentions)
Ru360, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Anti tom20, by Proteintech (2 mentions)
Fk506, by Cayman Chemical (2 mentions)
31 protocols using fluorescent labeled secondary antibody
1
Immunohistochemistry Protocol for Paraffin Sections
2
Immunofluorescence Staining of ACE-1 in BEAS-2B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B cells were incubated with polyclonal antisera against ACE-1 (catalog ab39172; Abcam). Secondary antibodies used were appropriate fluorescent labeled secondary antibodies (1:1000, Life Technologies, Inc.) as well as the nuclear marker DAPI (Biolegend). The slides were then mounted using Gelvatol (Sigma-Aldrich) solution prior to imaging using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany) under appropriate filter sets.
3
Multimodal Immunohistochemistry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded slides were utilized for immunostaining. Briefly, deparaffinized sections were stained with anti-ANG2, anti-F4/80 and anti-perilipin primary antibodies and incubated overnight. Then the corresponding fluorescent labeled secondary antibodies (Life Technologies, Carlsbad, CA) were added and finally the sections were counterstained with DAPI. The pimonidazole hypoxia probe staining was conducted according to the protocol of a commercially available kit (Hypoxyprobe-1 Plus Kit, Hypoxyprobe, Inc., Burlington, MA), followed by DAB staining. The fluorescence or histochemistry pictures were acquired with an Olympus FSX100 Microscope and analyzed by the Image Pro Plus software (Media Cybernetics, Rockville, MD).
4
Immunostaining of Paraffin-Embedded Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded slides were prepared by the University of Texas Southwestern Medical Center Histology Core for immunostaining. Briefly, deparaffinized sections were stained with anti-APP (1:200), anti-TIM23 (1:500) or anti-perilipin-1 (1:500) primary antibodies and incubated overnight at 4 °C. Then the corresponding fluorescent labeled secondary antibodies (Life Technologies, Carlsbad, CA, USA) were added and finally the sections were counterstained with DAPI before adding coverslips. The fluorescence images were acquired with an Olympus FSX100 Microscope.
5
Immunostaining of Paraffin-Embedded Samples
6
Cochlear Immunofluorescence of Hair and Supporting Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four days after injection, mice were sacrificed and cochleae were harvested by standard protocols [1 (link), 17 (link)]. For whole-mount immunofluorescence, primary antibodies against HC (MYO7A, #25-6790, Proteus Biosciences) and SC (SOX2, #sc-17320, Santa Cruz Biotech) markers and fluorescent-labeled secondary antibodies (Invitrogen) were used following a previously described protocol [1 (link)]. To quantify the proportion of GFP positive cells after Ad injection, we counted the number of GFP positive IHCs, OHCs, and SCs, which were then divided by the total number of IHCs, OHCs, and SCs, respectively, in a region spanning 200 µm in the apical, middle, or basal turn of the cochlea.
7
Immunofluorescence Microscopy of EB Spheroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EB spheroids for immunofluorescence microscopy were grown on coverslips prior to the experiment (1 EB spheroid per coverslip). EBs were fixed with 2% paraformaldehyde in PBS for 10 min and then were washed two times with DPBS at room temperature. Fixed cells were permeabilized with 0.5% Triton X-100 in PBS (PBS-T) for 5 min at room temperature. Blocking was performed with 10% FBS in 0.1% PBS-T for 30 min. For staining, cells were incubated with primary antibodies for 4 h at room temperature, washed four times with 0.1% PBS-T, and incubated for 1 h with fluorescent labeled secondary antibodies (Invitrogen). Cells were washed and mounted by VECTASHIELD (H-1200, Vector Laboratories) with DAPI (Sigma). Fluorescence was visualized on a Zeiss LSM700 confocal microscope (Carl Zeiss).
8
Fluorescence Immunohistochemistry of Neural Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections were washed with 1× (0.01 M) PBS and incubated with the primary antibodies at 4 °C overnight after 1 h blocking by 5% normal goat serum (NGS, Sigma) and 0.3% Triton X-100 (Sigma). The sections were then incubated at room temperature for 1 h with fluorescent-labeled secondary antibodies (Invitrogen) and washed with 0.01 M PBS 3 times before being observed under a confocal laser scanning microscope (STELLARIS 5, Leica Corp., Wetzlar, Germany). Fluorescence immunohistochemistry was performed using the following primary antibodies: rabbit anti-NeuN (Abcam177487, 1:500), mouse anti-Beta III Tubulin (Tuj1, MAB5564, 1:500), rat anti-Nestin (BDbiosciences, 556309, 1:500), goat anti-Foxj1 (AF3619, 1:500), mouse anti-Sox2 (Abcam79351, 1:500), mouse anti-GFAP (Abcam360, 1:500), F-actin (stain 488, Servicebio, CR2203082, 1:300), mouse anti-Alpha Tubulin (Servicebio, GB12200, 1:1000), goat anti-Sox9 (AF3075, 1:500), mouse anti-Gamma tubulin (Sigma, T6557, 1:500), rabbit anti-Arl13b (Proteintech, 17711-1-AP, 1:500), rabbit anti-Sox10 (Abcam155279, 1:500), rabbit anti-Olig2 (Abcam109186), rabbit anti-CD68 (Abcam125212, 1:500), goat anti-IBA1 (Abcam5076, 1:500), rat anti-MBP (Abcam7349, 1:500), goat anti-PDGFRA (AF307-NA, 1:50), and rat anti-BrdU (ab6326). Cell nuclei were stained with DAPI (Invitrogen).
9
Antibody Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit anti–TIMP-1 polyclonal antibody (pAb) was purchased from Abcam (Cambridge, USA). Mouse anti–CD82 monoclonal antibody (mAb), anti–GFP mAb, anti–α-tubulin mAb, and anti–β-Actin mAb were purchased from Santa Cruz Biotechnology. Stabilized Streptavidin-HRP Conjugate was obtained from Thermo Scientific. We purchased FITC-labeled phalloidin from Sigma (St. Louis, USA). We obtained horseradish peroxidase-conjugated secondary antibodies from Amersham Pharmacia Biotech (Piscataway, USA). Fluorescent-labeled secondary antibodies were purchased from Invitrogen.
10
Diaphragm Muscle Fiber Typing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serial cryosections were cut from the frozen biopsies (8 μm thick). Microscope slides were rehydrated in phosphate buffer (PBS) and subsequently blocked with phosphate buffer containing 1% bovine serum albumin (PBS-1%BSA). Cryosections were incubated with antibody for MyHCslow (1:100, ab11083, Abcam) and for MyHCfast (1:100, ab51263,Abcam) followed by appropriate fluorescent-labeled secondary antibodies (Invitrogen). Fibers were visualized by an antibody reactive to laminin (1:100, ab11575, Abcam). Following each incubation, cryosections were washed four times for 5 min with PBS. The cross-sectional area (CSA) of diaphragm muscle fiber was determined from a sample of 25–30 fibers of each type per animal (eight animals per group). Sections were analyzed with a Leica DM6000B microscope (Leica Application Suite). CSA was calculated by ImageJ software.
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!