Tumor tissues frozen in O.C.T. compound were cut into sections with 5 μm thickness. After blocking with 10% goat serum/1% BSA for 1 h, CAFs were stained with rabbit anti-α-SMA antibody (CST, 1:500) overnight at 4°C, and were then incubated with Alexa Fluor 488- or Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody at R.T. for 1 h. For immunofluorescent co-staining of α-SMA and CaMKK2, tumor tissue sections were first stained with goat anti-α-SMA antibody (Novus Biologicals, NB300–978, 1:50) overnight at 4°C and Alexa Fluor 488-conjugated donkey anti-goat secondary antibody; afterwards, sections were stained with rabbit anti-CaMKK2 antibody (Proteintech, 1:100) for 2 h at R.T., followed by 1 h incubation of Alexa Fluor 555-conjugated goat anti-rabbit secondary antibody. For immunofluorescent co-staining of CK8 and ARHGEF2, tumor sections were stained first with rat anti-CK8 antibody (TROMO-I, DSHB, AB_531826, 1:400) and subsequently stained with rabbit anti-ARHGEF2 antibody (Abcam, ab155785, 1:100), as described above; and the sequential tumor sections were stained with rabbit anti-α-SMA (CST, 1:500) at the same time. Images were captured at 40x magnification using the EVOS FL Cell Imaging System and analyzed using Fiji software.
Nb300 978
Manufactured by Novus Biologicals
Sourced in United States
The NB300-978 is a laboratory equipment product. It is a centrifuge tube designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab155785, by Abcam (1 mentions)
Fv3000, by Olympus (1 mentions)
Dl 1178, by Vector Laboratories (1 mentions)
L9393, by Merck Group (1 mentions)
Gtx632604, by GeneTex (1 mentions)
Ab234297, by Abcam (1 mentions)
Isolectin gs ib4 alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using nb300 978
1
Immunofluorescent Staining of Tumor Tissue Sections
2
Renal Biopsy Immunostaining Protocol
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After deparaffinization, antigen activation and blocking, the renal biopsy specimens were stained with primary antibodies for BAFF (LS-B2081, LSBio, WA, USA), CD68 (375602, Biolegend, CA, USA) and αSMA (NB300-978, Novus Biologicals, CO, USA) and secondary antibodies (see Supplementary Data S1 , available at Rheumatology online). The specimens were observed by confocal microscopy (Olympus FV3000, Tokyo, Japan).
3
Immunohistochemistry of Brain Slices
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Immunohistochemistry of the brain slices was performed as previously described (Foidl et al. 2018 (link)). At the end of a 2 or 8 week culturing period, the organotypic brain slices were fixed with 4% paraformaldehyde for 3 h and washed 3 × with 10 mM PBS. Slices were incubated for 30 min in 0.1% Triton-PBS (T-PBS) at room temperature (RT) and washed 3 × with PBS. Slices were blocked in 20% horse serum/0.2% bovine serum albumin (BSA)/T-PBS for 30 min at RT and subsequently incubated with primary antibody against laminin (1:500, Sigma, L9393) in T-PBS/0.2% BSA at 4 °C for 48 h. After subsequent washing 3 × with PBS, slices were incubated with Alexa488 conjugated anti-rabbit secondary antibody for 1 h and counterstained with DAPI. Slices were washed and mounted in mowiol. Some slices were additionally double-stained with primary antibodies against α-smooth muscle actin (1:1000, Novus Biologicals, NB300-978) followed by Alexa546 conjugated anti-goat secondary antibody or with tomato lectin Alexa649 (1:50, Vector Laboratories, DL-1178).
4
Antibody Panel for Tissue Characterization
5
Immunostaining of GLP-1 Receptor and Intima
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We performed immunostaining using anti-GLP-1 receptor antibody (NLS1205; NOVUS Biologicals, Minneapolis, MN, USA). The sections in citrate buffer solution were heated for 15 min at 90°C in a microwave oven for antigen retrieval. After washing in phosphatebuffered saline with Tween-20 (PBST), sections were blocked with 5% donkey serum for 60 min. And then, the sections were incubated with anti-GLP-1 receptor antibody alone or with anti-α-smooth muscle actin antibody (NB300-978; NOVUS) at 25°C for 14 h. After rinsing with PBST, second antibody (Alexa Fluor 488 donkey anti-rabbit IgG only or with Alexa Fluor 594 donkey anti-goat IgG; Invitrogen Corporation, Carlsbad, CA, USA) was added to sections. To perform immunostaining of the intima, isolectin GS-IB4 Alexa Fluor 594 conjugate (Invitrogen Corporation) was added to the artery sections. After rinsing with PBST, 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was added for 5 min at 25°C. TCF7L2 staining was performed using a previously reported method. And according to the previously established method, 12
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