Mice were euthanized and lumbar spinal cords were dissected under the level of T12 (using the last rib as a marker) and fixed in 4% paraformaldehyde (PFA) in PBS overnight. The samples were incubated in 30% sucrose in PBS for 24 h and then mounted in OCT. Using a cryostat, an initial 500 µm of each spinal cord was trimmed. Then, six transverse sections (thickness of 10 µm) with an interval of 100 µm were mounted on one slide, spanning a region of about 500 µm of mouse lumbar spinal cord at the level of L1-L2. The slides were air dried at room temperature and then stained as follows. Samples were permeabilized in 0.3% TritonX-100 in PBS for 30 min, then blocked in 1X Power Block (BioGenex, Fremont, CA) for 10 min at room temperature. Samples were incubated with a goat anti-ChAT antibody (EMD Millipore, Darmstadt, Germany) in 1% BSA and 0.3% TritonX-100 in PBS for 48 h at 4 °C. Samples were incubated with Alexa Fluor 555 donkey anti-goat IgG (Life Technologies, Carlsbad, California) for 2 h at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Using a Zeiss Ax10 microscope (Plan-APOCHROMAT 20X/0.8 Ph2 lens) equipped with an Axiocam HRM camera, images were taken from ventral horn areas of all spinal cord sections. Motor neuron cell bodies were counted using ImageJ software.
Alexa fluor 555 donkey anti goat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 donkey anti-goat IgG is a fluorescent secondary antibody conjugate. It is designed to detect and visualize goat primary antibodies in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 donkey anti mouse igg, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 647 donkey anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Rabbit anti sox9 antibody, by Merck Group (3 mentions)
Alexa fluor 546 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Ab243041, by Abcam (2 mentions)
Ab32010, by Abcam (2 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (2 mentions)
Gfp 1010, by Merck Group (2 mentions)
Ab5511, by Proteintech (2 mentions)
Ab10518, by LifeSpan BioSciences (2 mentions)
Ls c340696, by Abcam (2 mentions)
Alexa fluor 488 donkey anti chicken igg, by Thermo Fisher Scientific (2 mentions)
Irdye 680rd donkey anti rabbit igg, by LI COR (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488 donkey anti rat igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (2 mentions)
29 protocols using alexa fluor 555 donkey anti goat igg
1
Quantifying Lumbar Spinal Cord Motor Neurons
2
Immunofluorescence Staining of hVFF Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hVFF were washed twice with phosphate buffered saline (PBS), fixed with methanol (− 20 °C), air dried for 30 min and stored at − 20 °C. 30 min prior to immunofluorescence staining cells were thawed and blocked with 3% BSA (Sigma Aldrich, St. Louis, MO, USA) for 1 h. Subsequently, cells were immune-labelled using the UltraVision LP Detection System (Thermo Scientific, Fremont, CA, USA). The following antibodies were diluted in antibody diluent (Dako, Glostrup, Denmark) and applied for 90 min at room temperature: ACTA-2 aka. α-Smooth muscle actin (α-SMA) (goat IgG, 20 mg/mL, LifeSpan BioSciences, Seattle, WA, USA); collagen-1 (mouse IgG, Dilution 1:1000, Sigma Aldrich, MO, USA, product no. C2456); fibronectin (rabbit IgG, 0.56 µg/mL, Proteintech, Chicago, IL, USA). Cells were washed three times with PBS followed by incubation with the secondary antibody (Alexa Fluor 555 Donkey Anti-goat IgG, 10 mg/mL; Alexa Fluor 488 Donkey Anti-mouse IgG, 10 mg/mL, Alexa Fluor 488 Donkey Anti-rabbit IgG, 10 mg/mL; all from Life Technologies, Carlsbad, CA, USA) and DAPI (Life Technologies, Carlsbad, CA, USA) for 45 min. Slides were washed again with PBS, mounted with ProLong Gold antifade reagent (Life Technologies, Gaithersburg, MD, USA) and observed with a Leica DM600B fluorescent microscope (Leica, Wetzlar, Germany) connected to an Olympus DP72 digital camera (Olympus, Tokyo, Japan).
3
Fluorescent Antibody Labeling Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4
Immunostaining of PDZD8 and AChT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal antibodies to PDZD8 were from Sigma-Aldrich, and guinea pig polyclonal antibodies to AChT were from Sigma-Aldrich. Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 555 donkey anti-goat IgG as well as LysoTracker Red were from Thermo Fisher Scientific. Hoechst 33342 and filipin were from Sigma-Aldrich. Digitonin was from Tokyo Chemical Industry.
5
HUVEC Immunofluorescence Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coverslips were coated with collagen (Sigma-Aldrich, St. Louis, MO), and HUVECs were grown to confluency on them. At the end of the experiment, cells were fixed with 4% Paraformaldehyde, blocked with 10% donkey serum (Jackson Immuno Research Inc., West Grove, PA) and permeabilized with 0.1% Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO). Afterwards the primary antibody incubated for 1 h at room temperature. In order to visualize the binding of the primary antibody, cells were further incubated with fluorescent secondary antibodies for 1 h. As a secondary antibody Alexa Fluor 555 donkey anti goat IgG (Thermo Fisher Scientific, Waltham, MA) was used. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO). Finally the coverslips were affixed with Aqua-Poly/Mount (Polysciences Inc., Eppelheim, Germany). The images were taken with a Leica DMI 6000B microscope and were obtained with the same gain and offset conditions.
6
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 10% FBS in PBS for 1 h. Samples were stained with primary antibodies for overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature with Alexa Fluor 488-donkey anti-rabbit IgG, Alexa Fluor 555-donkey anti-goat IgG, Alexa Fluor 647-donkey anti-mouse IgG, Alexa Fluor 488-donkey anti-goat IgG and Alexa Fluor 555-donkey anti-mouse IgG (ThermoFisher Scientific). Hoechst staining was performed for 2 minutes at room temperature with Hoechst solution (ThermoFisher scientific, 62249). Images were taken using Leica SP8 con-focal microscope or Olympus IX71 fluorescence microscope.
7
Immunoblot and Immunohistochemistry Antibody Protocols
8
Immunoblot and Immunohistochemistry Antibody Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used for either immunoblots (IB) or immunohistochemistry (IHC) experiments: chicken antiGFP (1:1,000, Aves Labs, GFP-1010), rabbit polyclonal anti-MOR (1:350, Millipore, AB5511), rabbit polyclonal anti-PDE1A (1:500, Proteintech, 12442–2-AP), rabbit polyclonal anti-PPP1R1B (1:1,000, Millipore, AB10518), goat anti-tdTomato (1:500, LifeSpan Biosciences, LS-C340696), mouse monoclonal anti-FOXP1 (1:500, Abcam, ab32010), rabbit polyclonal anti-FOXP1 (IHC:1:1,000, IB: 1:5,000 (Spiteri et al., 2007 (link)), rabbit polyclonal anti-Calbindin (1:500, Millipore AB1778 and 1:250, Swant CB-38a for DEG quantification), goat anti-FOXP2 (N-terminal) (1:500, Santa Cruz 21069), rabbit polyclonal anti-b-Tubulin (IB: 1:10,000, Abcam, ab243041), and mouse monoclonal anti-SOX4 (1:500, Abcam, ab243041). All IHC following secondary antibodies were used at a 1:1,000 dilutions Alexa Fluor 488 Donkey Anti-Chicken IgG (Thermo Fisher, 703–545-155), Alexa Fluor 555 Donkey Anti-Goat IgG (Thermo Fisher, A-21432), Alexa Fluor 647 Donkey Anti-Rabbit IgG (Thermo Fisher, 711–605-152), Alexa Fluor 647 Donkey Anti-Mouse IgG (Thermo Fisher, A-31571). For IB, the following secondary antibodies were used at a 1:10,000 dilution: IRDye 800CW Donkey anti-Rabbit IgG (Li-Cor, 925–32213) and IRDye 680RD Donkey anti-Rabbit IgG (Li-Cor, 925–68071).
9
Dual-Receptor Immunofluorescence in Brain Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain coronal sections (thickness: 10 µm) were fixed in 4% paraformaldehyde in PBS for 10 min and rinsed in PBS (3 times for 5 min each) incubated in blocking solution (5% normal donkey serum in PBS) for 30 min at RT, then overnight at 4 °C with a mixture of primary antibodies. Final concentration of anti-5-HT1A receptor antibody (goat polyclonal, ab101914, Abcam) was 1:200, and of the anti-mGlu4 receptor antibody (rabbit polyclonal, SAB4501322, Sigma Aldrich) it was 1:50. On the following day, the sections were incubated for 1 h at room temperature with secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 555 donkey anti-goat IgG (Thermo Fisher Scientific), then washed 3 times with PBS and mounted using DAPI-containing mounting medium (Sigma Aldrich). Images were acquired using an Axio Imager 2 fluorescence microscope (Carl Zeiss MicroImaging GmbH, Germany) equipped with excitation and emission filters compatible with the fluorophores.
10
Immunohistochemical Analysis of Neurogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde. The brains were excised and postfixed, then cut using a cryotome into serial coronal sections (25 µm thick) (Leica CM 1850; Leica, Wetzler, Germany). Sections were incubated in a blocking solution (10% normal donkey serum, 0.3% Triton X-100 in PBS, 2.5% bovine serum albumin in PBS) at room temperature for 2 h. For DCX staining, the sections were washed and incubated for 12 h at 4°C with goat anti-DCX antibody (diluted 1:125; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by washing and incubation for 2 h in secondary antibody (diluted 1:1,000; Alexa Fluor 555 donkey anti-goat IgG; Invitrogen Life Technologies, Carlsbad, CA, USA). For AnkG/DCX double staining, sections were incubated in mouse anti-AnkG antibody (diluted 1:50; Merck Millipore, Darmstadt, Germany) and goat anti-DCX antibody (diluted 1:125; Santa Cruz Biotechnology, Inc.) at 4°C for 48 h. Sections were rinsed and incubated with Alexa Fluor 488 donkey anti-mouse IgG + Alexa Fluor 555 donkey anti-goat IgG (all diluted 1:1,000; Invitrogen Life Technologies). The sections were then washed and mounted with Vectashield mounting media.
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!