Sentences with phrase «primary antibodies»

Primary antibodies against GCTM - 2 and TG30 were detected using goat anti-mouse IgM - AF647 and goat anti-mouse IgG2a - AF488, respectively (Invitrogen, Carlsbad, CA).

Listing of primary antibodies used together with their dilutions, sources, and molecular weights.

Primary antibodies were incubated overnight at 4 °C, washed with PBS and then incubated with the secondary antibodies at 37 °C for 1 hour.

Subsequently, the primary antibodies were added to PBS and incubated overnight at 4 °C with gentle shaking.

Primary antibodies included: SP1 - ER - α (RM -9101-SO, Thermo Fisher Scientific), Cleaved N1 - ICD (100-401-407, Rockland), Jagged1 (Santa Cruz), Actin (Santa Cruz, sc - 1616), and HIF - 1α (610959, BD Biosciences).

Cells on dishes, or sections were blocked and incubated with appropriate combinations of primary antibodies as described previously [9].

For colocalization of both tracers or one tracer together with ZENK signals, primary antibodies (Egr - 1 / ZENK, 1 ∶ 500; CtB, 1 ∶ 300 in PBS - T) were detected by an appropriate secondary antibody (polyclonal goat raised against rabbit IgG labeled with fluorescent dyes Alexa488; Molecular Probes Europe BV, Leiden, The Netherlands, 1 ∶ 400 in PBS - T).

Membranes were then incubated with the same blocking solution containing rabbit polyclonal primary antibodies against HIF - 2α (1:500, ab199).

The following primary antibodies were used: Oct4 (BD Transduction Laboratories, 1 ∶ 1000), phospho - p38 MAPK and total p38 MAPK (Cell Signaling), β - Actin clone AC - 15 (Sigma Aldrich, 1 ∶ 5000) and GAPDH (Ambion, 1 ∶ 40,000).

The following primary antibodies were used: Oct - 4 (19857 Abcam) 1 ∶ 1000; β - III tubulin clone SDL.3 D10 (T8660 Sigma) 1 ∶ 500; Nestin (611658 BD Transduction laboratories) 1 ∶ 500 and TH (P40101 Pel - Freez) 1 ∶ 500, and as secondary antibodies: Alexa Fluor 594 Goat Anti-Mouse, Alexa Fluor 488 Goat Anti-Rabbit, Alexa Fluor 594 Goat Anti-Rabbit.

Membranes were blocked for 2 hours in 10 % milk in PBS - 0.05 % Tween - 20 and incubated overnight in 10 % milk containing primary antibodies raised in Mouse: RPE65 (1 ∶ 2000, Chemicon); RLBP1 (CRALBP, 1 ∶ 2000, Affinity BioReagents, CO, USA); KRT8 (1 ∶ 2000, Chemicon); PEDF (1 ∶ 500, Chemicon); PMEL17 (1 ∶ 100, Dako); Rabbit: MERTK (1 ∶ 500, Abcam); FAK (0.1 µg / ml, Stratech) and MITF (1 ∶ 500, Chemicon).

Table 1 lists the primary antibodies that were used.

Alexa Fluor 488 - and / or 594 - conjugated secondary antibodies (1:500; Invitrogen) reactive to the species of the primary antibodies, and the Olympus IX70 inverted microscope, were used for visualization.

It's best to select primary antibodies raised in different species, or at least from different IgG subclasses, so that secondary antibodies exclusively recognize only one primary antibody.

The membranes were incubated with primary antibodies against p - Smad1 / 5 (1:1000), Smad1 / 5 (1:1000; Abcam) or β - actin (1:2000) overnight at 4 °C, washed, incubated with the appropriate goat IR700 / 800 - labelled secondary antibodies (1:10000; LICOR), washed and visualized with Odyssey (LICOR).

Other primary antibodies were from Santa Cruz Biotechnology (STAT1, STAT2, IRF - 9, and IRF - 1), Sigma (β - actin), and Cell Signaling (pTyr701 - STAT1 and pTyr690 - STAT2).

Looking for monoclonal primary antibodies to use with the secondary nanobody toolbox?

This process can be avoided direct antibody fluorophore conjugates are used, but these are usually more expensive than unconjugated primary antibodies.

Primary antibodies specific to Oct4 and Nanog were detected using Alexa 568 conjugated secondary antibodies (red).

Instead, you can pre-incubate individual primary antibodies with secondary nanobodies conjugated with the desired fluorophores.

Primary antibodies used were mouse anti-Oct3 / 4 (Santa Cruz) and rabbit anti-Nanog peptide specific antibodies (a gift from Ian Chambers)[8].

Primary antibodies utilized are listed in Table S1.

The cells were incubated with the primary antibodies, polyclonal goat anti-human Nanog at a dilution of 1:200 and monoclonal mouse anti-human SSEA - 1 at 1:200 (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), overnight at 4 °C.

Rabbit anti-CCHa2 (1:1,000)[19], rabbit anti-Dilp2 (1:2,500; T. Ida), rabbit anti-Dilp2 (1:2,000, a gift from T. Nishimura, RIKEN Center for Developmental Biology)[27], rabbit anti-Dilp5 (1:2000, a gift from T. Nishimura)[27], mouse anti-Repo c8D12 (1:100; DSHB), mouse anti-GFP (1:500; Invitrogen), and rabbit anti-NPF (1:500; Ray Biotech, Inc) were used as primary antibodies, with anti-mouse-Alexa 488 (1:500; Invitrogen), anti-mouse-Cy3 (1:500; Jackson ImmunoResearch), anti-rabbit-Alexa 488 (1:500; Invitrogen), and anti-rabbit-Cy3 (1:500; Jackson ImmunoResearch) used as secondary antibodies.

Information regarding the primary antibodies utilized in immunofluorescence analysis and primers utilized in RT - PCR, genotyping, and bisulfite sequencing analyses.

Tip 6: For detection of immunoprecipitated proteins by Western blotting, without or reduced detection of non-specific artifacts (such as the heavy and light chains of the immunoprecipitating antibody), detect primary antibodies using HRP - conjugated anti-rabbit light chain - specific (L) antibody and HRP - conjugated Protein A instead of traditional HRP - conjugated secondary antibodies.

Antisera against IgG (H+L) or against the F (ab») 2 fragments of IgG will also bridge PAP to IgM primary antibodies (such as IgM monoclonal antibodies) by virtue of common light - chain recognition.

For double labeling, the cells were incubated with primary antibodies against TuJ1 and L1 or GFAP, then biotinylated anti-mouse or rabbit (rat) IgG (Vector, Burlingame, CA, USA), and finally with avidin - rhodamine (Vector) and corresponding FITC - conjugated secondary antibodies (from different host species, Chemicon and Molecular Probes, Invitrogen).

Omission of primary antibodies resulted in no detectable fluorescent staining (data not shown).

Briefly, 1 mL cell suspension (1 × 106 cells) was incubated for few minutes in rat specific Fc block solution (BD Biosciences, San Jose, CA, USA) followed by incubation with fluorescence - tagged primary antibodies - CD11b - FITC (BD Biosciences), TNF - α - PE (BD Biosciences) and NF - κB - FITC (in 1:100 dilution) at 4 °C for 30 min in dark.

Blocking was done in 10 % NGS in PBS - T during 1 h. Primary antibodies were incubated overnight in 5 % NGS at 4 °C.

The primary antibodies were used at a dilution of 1:1,000 unless otherwise stated and their sources and molecular weights were as follows: Cell Signaling Technology (Danvers, MA, USA): β - actin, monoclonal HRP conjugate, 45 kDa; CREB, monoclonal, 43 kDa.

For the detection of specific antigens, the following primary antibodies were used: mouse monoclonal anti-Tuj1 (Covance, Princeton, NJ, USA) as a neuronal marker, rabbit anti-L1 (kindly provided from Dr. Asou)[52] and rat monoclonal anti-L1 (Millipore, Billerica, MA, USA) as granule cell markers, rabbit anti-calbindin D28K (Millipore) as a Purkinje cell marker, and rabbit anti-glial fibrillary acidic protein (GFAP, DAKO, Denmark) as an astrocyte marker.

This was followed by overnight incubation with primary antibodies for IL - 6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), IL - 1β (Santa Cruz Biotechnology Inc.), TNF (BD PharMingen, San Jose, CA, USA), collagen (Santa Cruz Biotechnology Inc.), and amylase (Santa Cruz Biotechnology Inc.) at a dilution of 1:100 in a humified chamber.

Generation, purification, and testing of primary antibodies Gold -, enzyme - and fluorescent labeling of secondary antibodies In situ hybridization Light - and electron microscopy

Whole antisera against IgG (H+L) are recommended for bridging PAP to primary antibodies.

Fab fragments can be used to block endogenous immunoglobulins on cells, tissue, or other surfaces, and to block the exposed immunoglobulins in multiple labeling experiments using primary antibodies from the same species.

However, the company's line is well - characterised, with high quality primary antibodies, some of which have been offered for thirty years.

This option is useful when the primary antibodies are from the same host (e.g., a mouse monoclonal against antigen A and a mouse monoclonal against antigen B).

Primary antibodies were omitted for negative controls.

Primary antibodies, Tie2 (R&D, #AF313, 1:100), Calpain1 (Sigma, #C5736, 1:100), FOXO3A (Cell Signaling, # 2497, 1:100), actin (phalloidin, Invitrogen, #A12379, 1:500) NG2 (Millipore, #Ab5220, 1:100), CD31 (BD Bioscience, # 553370, 1:100) and Mac - 1 (BD Bioscience, # 550282, 1:50) were prepared in 1 % BSA / PBS containing 0.3 % Triton - X 100 and incubated overnight at 4 °C.

Beginning in October 2017, Bio-Techne began to distribute Vector Laboratories products to complement the over 40,000 primary antibodies from Novus Biologicals ® validated for immunocytochemistry and immunohistochemistry.

Membranes were probed with the following primary antibodies diluted in TBS - T with 5 % NFM overnight at 4 °C: calsequestrin rabbit polyclonal antibody (1:2,000 dilution, ab3516, Abcam), PPARδ rabbit polyclonal antibody (2 μg • ml — 1 dilution, ab23673, Abcam), COX polypeptide Va mouse monoclonal antibody (1:1,000 dilution, MS409, MitoSciences), phosphorylated Smad2 (1:1,000 dilution, 3101, Cell Signaling Technology), Smad2 / 3 (1:1,000 dilution, 3102, Cell Signaling Technology), GAPDH rabbit polyclonal antibody (1:1,000 dilution, ab9485, Abcam).

Examining the gill microbiota, they found that IgT was the primary antibody coating bacteria in the gills, consistent with the team's earlier findings in fish skin and gut.

This staining is specific to the primary antibody for it is not present in sections in which the primary antibody was omitted (B) a secondary only control.

The slides were incubated with the primary antibody overnight at 4 °C, rinsed three times with PBS, incubated for 10 min in protein blocking solution, and incubated with the appropriate secondary reagent.

Since alkaline phosphatase, coupled to the secondary antibody for the BrdU primary antibody, may have complicated the interpretation of the wounding experiments, we also treated human corneal - limbal tissues with BrdU histochemical assays in which fluorescein, rather than alkaline phosphatase, was coupled to the secondary antibody.

The sample was incubated with the primary antibody (1/100 in PBS) for 15 minutes at room temperature.

The sample was incubated with the primary antibody (1/100 in 1x HBSS + 0.02 % Triton X-100 + 1.5 % FBS) for 3 hours at 25 °C.

The fixed tissues were covered on the endothelial side with a 1:1000 dilution of anti-BrdU primary antibody.