Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 20% normal goat serum and permeabilization for 10min with 0.2% Triton X-100 in PBS, cultures were incubated overnight at 4°C with the following antibodies (mAb, monoclonal; pAb, polyclonal): Nestin ( MAB5326, Millipore, 1:200), Ki67 (ab16667, Abcam, 1:1000), Oct4 (MAB4401, Millipore, 1:2000), Pax6 (Developmental Studies Hybridoma Bank; 1:30), β-tubulin isotype III (β-tubIII, mAb, MMS-435P Covance, 1:400), Glial Fibrillary Acidic Protein (GFAP, z033429-2, Dako, 1:400), Lamp1 (pAb, ab24170, Abcam, 1:750), Cleaved Caspase-3 (pAb, #9661, Cell Signaling, 1:500), NLRP3 (pAb, AG-20B-0014-C100, AdipoGen, 1:200), Microtubule-Associated Protein 2 (MAP2, AB5622, Millipore, 1:400), Caspase 1 (Casp1, SC-515, Santa Cruz Biotechnology, 1:100), LC3B (pAb#2775, Cell Signaling, 1:400), GAPDH (ab9485, Abcam, 1:3000). After removal of the primary antibodies and repeated washes with PBS, cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 633 or 488 (anti-mouse and/or anti-rabbit, Molecular Probes). Samples were then labeled with DAPI (0.3 μg/ml, Roche) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a confocal microscope.
Z033429 2
Manufactured by Agilent Technologies
Sourced in Canada
The Z033429-2 is a laboratory equipment product manufactured by Agilent Technologies. It is a precision instrument designed for analytical measurements in various scientific and research applications. The core function of this product is to provide accurate and reliable data for analysis purposes. Further details on the specific intended use or features of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Ab154769, by Abcam (2 mentions)
Coated chamber slides, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 goat anti rabbit, by Abcam (2 mentions)
Goat anti rabbit alexa fluor 568 antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Leica sp5 system, by Leica camera (2 mentions)
Fluorsave reagent, by Merck Group (2 mentions)
Ab24170, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ag 20b 0014 c100, by Adipogen (1 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
Mab5326, by Merck Group (1 mentions)
Sc 515, by Santa Cruz Biotechnology (1 mentions)
Mab4401, by Merck Group (1 mentions)
Ab5622, by Merck Group (1 mentions)
Mms 435p, by Fortrea (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Ab9485, by Abcam (1 mentions)
10 protocols using z033429 2
1
Immunofluorescence Analysis of Neural Progenitor Cells
2
Comprehensive Neurological Protein Analysis
3
Analyzing Parkinson's Disease Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human α‐Syn (s7820), Rapa (V900930) and the antibodies against p62 (P0067), Atg5 (A2859), TH (T1299), and Actin (A3854) were purchased from Sigma‐Aldrich. Antibodies against phosphorylated and total MAP kinases, Akt, mTOR, and p70S6K were from Cell Signaling Technology (CST). Other primary antibodies included: LC3 (NB100‐2220, NOVUS), Iba1 (019–19741, WAKO), p‐S129 α‐Syn (ab51253, Abcam), MJFR1 clone anti‐α‐Syn (ab138501, Abcam) for western blotting and LB509 clone anti‐α‐Syn (180215, Thermo) for immunostaining, DAT (MAB369, Millipore), NeuN (MAB377, Millipore), GFAP (z033429‐2, DAKO), phosphorylated FOXO3 (AP0684) and FOXO3 (A0102, ABclonal). The inhibitor for p38 (SB202190) and PI3K (Wort) was purchased from Selleck, BafA1 from Abcam, and CHX from Med Chem Express.
4
Visualizing Chromatin-Associated Proteins in Cells
5
Visualizing Chromatin-Associated Proteins in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For GFP visualization cells were grown on poly-L-lysine (Sigma Aldrich) coated chamber slides (ThermoFisher Scientific), fixed in 4% formaldehyde (FA) and nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).
6
GFAP Immunofluorescence Staining and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serial free-floating sections were stained with rabbit anti-GFAP antibody as previously [31] (link). Sections were washed three times with PBS and blocked for 60 min using 3% normal goat serum in PBS with 0.2 Triton-X for permeabilization. Sections were incubated in anti-GFAP antibody (Z033429–2, Agilent; 1:1000) for 3 h. Following three 10 min PBS washes, sections were incubated with goat anti-rabbit 594-labeled antibody (DI-1594, Vector Biolabs; 1:250) for 1 h. Following three 10 min PBS washes, sections were mounted on slides. All staining steps were performed at room temperature on a shaker.
Imaging was performed with the Nikon Eclipse 80i microscope (40X total magnification) and MetaMorph 7.0 software (Molecular Devices) was used to define the percent area coverage for X-34 and GFAP staining.
Imaging was performed with the Nikon Eclipse 80i microscope (40X total magnification) and MetaMorph 7.0 software (Molecular Devices) was used to define the percent area coverage for X-34 and GFAP staining.
7
Perfusion and Brain Tissue Imaging
8
Hematoxylin and Eosin Staining of Trigeminal Ganglion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hematoxylin and Eosin (H&E) staining was conducted on frozen TG tissue sections (5μm) to visualize ganglion structure. Briefly, slides were incubated in Harris’ Hematoxylin for 5 min, rinsed in H20 for 5 min and differentiated in acid alcohol (1% HCl in 70% ethanol) for 10 sec. Next, sections were rinsed with distilled H20 (dH20) for 5 min, incubated in ammonia water (0.25% ammonia hydroxide in dH20) for 3 min and rinsed with dH20 2X’s (5 min). Sections were dipped in alcoholic Eosin for 10 sec, rinsed with dH20 2X’s (5min), dehydrated in ethanol and then mounted onto coverslips (Permount, Fisher Scientifc; Hampton, NH).
Protein expression was also determined in frozen TG sections using an antibody directed against glial fibrillary associated protein (GFAP) (Z033429-2, Agilent Technologies, Santa Clara, CA). A standard immunohistochemical avidin-biotin-peroxidase complex technique (Vectastain Elite ABC kit, PK-6100, Vector Laboratories, Burlingame, CA) was used to visualize protein expression using 0.02% of 3-diaminobenzidine tetrahydrochloride. Sections were then counterstained with methyl green. Images were acquired with an Olympus BX3 microscope equipped with a DP73 17.28 megapixel digital color camera using a 20X objective (Olympus Life Science; Center Valley, PA).
Protein expression was also determined in frozen TG sections using an antibody directed against glial fibrillary associated protein (GFAP) (Z033429-2, Agilent Technologies, Santa Clara, CA). A standard immunohistochemical avidin-biotin-peroxidase complex technique (Vectastain Elite ABC kit, PK-6100, Vector Laboratories, Burlingame, CA) was used to visualize protein expression using 0.02% of 3-diaminobenzidine tetrahydrochloride. Sections were then counterstained with methyl green. Images were acquired with an Olympus BX3 microscope equipped with a DP73 17.28 megapixel digital color camera using a 20X objective (Olympus Life Science; Center Valley, PA).
9
Immunofluorescent Labeling of Glial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 2% paraformaldehyde and then permeabilized in 0.5% Triton X-100 before probing overnight at 4 °C with anti-GFAP (1:1000, Agilent Technologies #Z033429-2) or anti-GalC hybridoma supernatant (1:4). Antibody binding was detected with appropriate Alexa Fluor-coupled antibodies at a concentration of 1 μg/ml (Thermo Fisher Scientific), applied for 1 h. All antibodies were diluted in HBSS (Invitrogen) plus 5% fetal calf serum with 0.5% sodium azide and 0.1% Triton X-100. Stained cells on coverslips were rinsed three times in 1 × PBS, counter-stained with 4′6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific #D1306) and mounted on glass slides with Fluoromount-G (SouthernBiotech #0100-01). Images were acquired by LAS AF software using a Leica TCS SP5 laser confocal microscope (Leica Microsystems, Mannheim, Germany).
10
Histopathological Analysis of Rat MCAO Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On day 60, rats were deeply anesthetized as described above for MCAO before being perfused with a 4% solution of paraformaldehyde in PBS. Rat brains were harvested, and cryosectioned. 25 μm thick coronal cryosections of the brain were mounted onto glass slides and stained for Cresyl Violet. Sections stained for cresyl violet were analyzed for differences in width of corpus callosum at bregma -0.26. Using ImageJ (NIH), pixel distance was standardized against a digital micrometer, and width was determined as the distance from the superior to inferior aspect of the CC at the midline. Moreover, sections were analyzed for immunohistological (IHC) staining with antibodies for Glial Fibrillary Acidic Protein (GFAP) (1:500; Agilent #Z033429-2), Ionized Calcium Binding Adaptor Molecule-1 (IBA-1) (1:500; Synaptic Systems #234308), Ki-67 (1:200; ThermoFisher #MA5-14520), and NeuN (1:200, ThermoFisher #PA5-78499). In five randomly selected stained cross sections, five ROIs (40×/section) were chosen that encircled the infarct on the ipsilateral side or was equivalently located on the contralateral side. Likewise, rats that had received MSCs loaded with QDs were similarly analyzed.
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!