Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Immunofluorescent analysis of 100% methanol, 0.1% TritonX-100 permeabilized K562 (Human chronic myelogenous leukemia lymphoblast cell line ) cells labelling Human Nucleoli with ab190710 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing nucleolar staining in K562 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/500 dilution (red).

Tubulin is detected with Anti-beta Tubulin rabbit monoclonal antibody (ab179513) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab190710 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary at 1/1000 dilution.
-ve control 2 : Anti-beta Tubulin rabbit monoclonal antibody (ab179513) at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Immunofluorescent analysis of 100% methanol, 0.1% TritonX-100 permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Human Nucleoli with ab190710 at 1/50 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing nucleolar staining in HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-beta Tubulin rabbit monoclonal antibody (ab179513) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/500 dilution (red).

The negative controls are as follows :
-ve control 1 : ab190710 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150080) secondary at 1/500 dilution.
-ve control 2 : Anti-beta Tubulin rabbit monoclonal antibody (ab179513) at 1/200 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) (ab150113) secondary at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Immunohistochemical analysis of paraffin-embedded sections human colon cancer tissue labelling Human Nucleoli with ab190710 at 1/2000 dilution, followed by secondary ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879). Nuclear staining in human colon cancer tissue is observed. Counter stained with Hematoxylin. Heat mediated antigen retrieval was performed using Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879).

The section was incubated with ab190710 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labelling Human Nucleoli with ab190710 at 1/1000 dilution, followed by secondary antibody the ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879). Nuclear staining in human kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use Goat Anti-Mouse IgG H&L (HRP polymer) (ab214879).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

The section was incubated with ab190710 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Flow cytometry overlay histogram of 4% paraformaldehyde-fixed, 90% methanol permeabilized K562 (Human chronic myelogenous leukemia lymphoblast) cells labelling Human Nucleoli with ab190710 at 1/1000 dilution (red) compared with a Mouse monoclonal IgG (black) and an unlabelled control (Cell without incubation with primary antibody and secondary antibody) (blue). Goat anti mouse IgG (Alexa Fluor^® 488) (ab150113), at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Exposure times :
lane 1 : 3 seconds
lane 2 : 5 seconds

5% NFDM/TBST

Lysates were made freshly and used in WB immediately to minimize protein degradation.

All lanes:

Western blot - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (ab190710) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 100 kDa

false

• IHC

CiteAb

Immunohistochemistry - Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker (AB190710)

Immunohistochemistry using Anti-Human Nucleoli antibody [NM95] - Nucleolar Marker, ab190710. Publication image from Han, Y. et al., 2022, Nat Commun, 35288560. Legend direct from paper.

Co-culture assay of TBBC and human mesenchymal stem cells (MSC).a Flow chart illustrating in vitro assay setup. TBBCs were pre-differentiated for 13 days on air-liquid interface prior to co-culture, on the other side, MSCs were pre-induced as pellets in chondrogenic medium or pre-induced as monolayers in osteogenic medium for 3 days before co-culture with TBBCs. RNA of MSC samples were collected every 5 days post-co-culture for PCR and fixed at the endpoint (Day 21) for staining. b Real-time PCR measurement of SOX9/ACAN (Chondrogenic-related) and RUNX2/BGLAP (Osteogenic-related) gene expressions on MSCs at D5, D10, and D15 post co-culture (n = 2 experimental replicates per group). w/nc denoting co-culture with non-TO control, w/TO denoting co-culture with TO-TBBC. Two-tailed Student t-tests were performed and p values are indicated to show statistical significance. c HE and Alcian Blue staining on MSC spheroids which were co-cultured with control or TO cells for 21 days, to reveal the progress of chondrogenesis (n = 2 independent experiments). MSC spheroids cultured alone under basic conditions and spheroids under standard differentiation conditions were examined in parallel as negative and positive controls. Scale bar, 100 µm. d, e Schematic diagrams showing two in vivo approaches to perform TBBC-MSC co-culture assay. Dual-cell transplantation onto immunodeficient NSG mice was performed either by subcutaneous injection of TBBCs (2 x 106 cells/spot) : MSC (2 x 106 cells/spot) mixture resuspended with 50% Growth factor-reduced Matrigel in a total volume of 200 µl/spot, or via minor surgery with aggregated MSC (106 cell/spheroid) surrounded by a gel-like layer of TBBCs resuspended in Matrigel. Cartoon mice in this graph were adapted from the online source clipartcraft.com. f HE and Alcian Blue staining on xenografts collected from approach D (n = 2 independent experiments). The human origin of growths found in nodules was confirmed by IHC staining with an anti-human nucleoli antibody. Scale bar, 200 µm. g HE and Alcian Blue staining on xenografts collected from approach E (n = 2 independent experiments). The human origin of growths was confirmed by IHC anti-human nucleoli staining. Scale bar, 200 µm. Source data are provided as a Source Data file.