White adipose tissue was excised, fixed in formalin overnight, embedded in paraffin and sectioned. The immunofluorescence analysis of netrin-1 (1:200, AF1109; R&D systems), caveolin-1 (1:500, 610059; BD bioscience), Unc5b (1:200, ab54430; Abcam), F4/80 (1:500, MCA497GA; Abd serotec), CD68 (1:500, MCA1957; Abd serotec) was conducted after deparaffinization as described previously61 (link). Secondary antibodies were then applied (Alexa Fluor-568, A11011; Alexa Fluor-488, A11006; 1:500 Invitrogen). Netrin-1 and Unc5b staining was amplified using the biotin conjugated antibodies (1:100, BA-2000 and BA-9500; Vector Laboratories) followed by streptavidin conjugated AMCA (1:200, Sa-5008; Vector Laboratories) staining. Sections were mounted and visualized using a Nikon Eclipse microscope.
Af1109
Manufactured by R&D Systems
Sourced in United States
AF1109 is a recombinant human protein that functions as a growth factor. It is produced in a murine cell line.
Automatically generated - may contain errors
Lab products found in correlation
Ab54430, by Abcam (2 mentions)
Ba 2000, by Vector Laboratories (2 mentions)
Mca1957, by Bio-Rad (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Sa 5008, by Vector Laboratories (2 mentions)
Ba 9500, by Vector Laboratories (2 mentions)
Mca497ga, by Bio-Rad (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Sc 6535, by Santa Cruz Biotechnology (1 mentions)
Img 5143a, by Novus Biologicals (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Goat anti dcc, by Santa Cruz Biotechnology (1 mentions)
Af 1079, by R&D Systems (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab134017, by Abcam (1 mentions)
A22287, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Hpa003956, by Merck Group (1 mentions)
Ab553, by Merck Group (1 mentions)
6 protocols using af1109
1
Immunofluorescence Analysis of Adipose Tissue
2
Detecting DCC and NTN1 expression
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Whole-cell protein extracts were prepared from N2A and U251 cells, 20 hr after transfection with pCAG-DCC:TDTOMATO and pCAG-myr-TDTOMATO constructs (1 µg) as previously described (Bunt et al., 2010 (link)). Moreover, COS-7 protein extracts were prepared 48 hr after transfection with the pCAG-DCC:TDTOMATO wildtype and missense mutant receptor constructs (0.2 µg). Western blots were performed to detect mouse DCC expression levels using a goat polyclonal anti-DCC antibody (1:200 COS-7 or 1:800, sc-6535, Santa Cruz Biotechnology) and mouse NTN1 using a goat polyclonal antibody (1:500 U251 or 1:1000 N2A, AF1109, R&D Systems). GADPH was used as a loading control and detected using rabbit monoclonal anti-GADPH antibodies (1:2000, 2118, Cell Signaling Technology for COS-7; 1:1000, IMG-5143A, IMGENEX for N2A and U251).
3
Anti-inflammatory effects of NTN-1 in DED
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To investigate the anti-inflammatory effects of NTN-1, DED mice were randomly categorized into four groups (five mice per group): the first three groups received 25 ng/5 µL recombinant mouse NTN-1 (1109-N1; R&D, Minneapolis, MN, USA), 250 ng/5 µL anti-mouse NTN-1 antibody (R&D, AF1109), or 250 ng/5 µL goat IgG (R&D, AB-108-C), and mice in the control group received PBS. The mice in each group received 5 µL of corresponding medication via ocular surface instillation four times daily (at 8:00 a.m., 11:00 a.m., 2:00 p.m., and 5:00 p.m.) for 5 consecutive days during the entire course of DED induction.
4
Immunofluorescence Analysis of Adipose Tissue
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White adipose tissue was excised, fixed in formalin overnight, embedded in paraffin and sectioned. The immunofluorescence analysis of netrin-1 (1:200, AF1109; R&D systems), caveolin-1 (1:500, 610059; BD bioscience), Unc5b (1:200, ab54430; Abcam), F4/80 (1:500, MCA497GA; Abd serotec), CD68 (1:500, MCA1957; Abd serotec) was conducted after deparaffinization as described previously61 (link). Secondary antibodies were then applied (Alexa Fluor-568, A11011; Alexa Fluor-488, A11006; 1:500 Invitrogen). Netrin-1 and Unc5b staining was amplified using the biotin conjugated antibodies (1:100, BA-2000 and BA-9500; Vector Laboratories) followed by streptavidin conjugated AMCA (1:200, Sa-5008; Vector Laboratories) staining. Sections were mounted and visualized using a Nikon Eclipse microscope.
5
E11.5 Head Protein Extraction
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E11.5 heads were lysed in 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1× Pefabloc SC PLUS protease inhibitor (Roche). Primary antibodies used include goat anti-Boc (1:1000; R&D, AF2385), goat anti-Dcc (1:1000; Santa Cruz, SC-6535), rabbit anti-Flk1 (1:2000; Cell Signaling; as described in Gu et al., 2003 (link)), goat anti-neogenin 1 (1:1000; R&D, AF1079) and rat anti-Ntn1 (1:500; R&D, AF1109). Each blot was performed twice using independent lysates.
6
Immunostaining of Neuronal Markers
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(1:250, ab610921, BD Biosciences), rat anti-Nestin (AB 2235915, DSHB), chicken anti-Nestin (1:1000, ab134017, Abcam), goat anti-Netrin1 (AF1109, R&D Systems), mouse anti-Neurofilament (1:500; MAB1621, Chemicon), rabbit anti-Nfia (1:500; ARP32714, Aviva Systems Biology), rabbit anti-Nfib (1:500; HPA003956, Sigma), rabbit anti-neuronal-specific-ßIII-tubulin (1:500; ab18207, Abcam), rabbit antiphospho p44/42 Mapk (or Erk1/2; 1:250; 9101, Cell Signaling), rabbit anti-Sox9
(1:500, AB553, Merck), and goat anti-TDTOMATO (1:500, ab8181-200, Sicgen). For actin staining, Alexa fluor-conjugated phalloidin (A22287, Thermofisher scientific) was incubated on tissue for thirty minutes in the dark as per the manufacturer's instructions, prior to the addition of primary antibodies. Immunohistochemistry was performed in a similar manner for cultured cells, with the following minor exceptions: HEK293T cells expressing wildtype and mutant pCAG-DCC:TDTOMATO constructs were not permeabilized to confirm exogenous DCC receptor localisation to the plasma membrane.
(1:500, AB553, Merck), and goat anti-TDTOMATO (1:500, ab8181-200, Sicgen). For actin staining, Alexa fluor-conjugated phalloidin (A22287, Thermofisher scientific) was incubated on tissue for thirty minutes in the dark as per the manufacturer's instructions, prior to the addition of primary antibodies. Immunohistochemistry was performed in a similar manner for cultured cells, with the following minor exceptions: HEK293T cells expressing wildtype and mutant pCAG-DCC:TDTOMATO constructs were not permeabilized to confirm exogenous DCC receptor localisation to the plasma membrane.
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