Differentiated WT or FcRn KO podocytes were fixed in 4% paraformaldehyde. After fixation, podocytes were rinsed with PBS, permeabilized in 0.1% Triton-X100 in PBS (PBS-X), blocked in 5% BSA in PBS-X and incubated overnight at 4°C with the following primary antibodies depending on the experiment: rat anti-mouse LAMP1 (1:500, Santa Cruz, cat. # sc-19992), goat anti-mouse IgG (1: 100, Vector Labs, cat. #NC0207995), rat anti-mouse IgG1 (1:100, Biolegend, cat. # 50169164), rabbit anti-mouse Rab11 (1:200, Cell Signaling, cat. # 5589), rabbit anti-mouse Rab7 (1:200, Cell Signaling, cat. #9367). Podocytes were rinsed and incubated with the appropriate secondary antibodies (Alexa 568 goat anti-rat, Alexa 488 goat anti-rat, Alexa 488 goat anti-rabbit, Alexa 568 donkey anti-goat, Alexa 568 goat anti-rabbit, Invitrogen). Hoechst was used to stain nuclei and Alexa 635 phalloidin was used to stain actin. Images were acquired using a Zeiss LSM 780 microscope. Colocalization between immune complexes and LAMP1 or ICs and Rab11 was evaluated using the Coloc2 Tool in ImageJ (NIH). Lysosomal area was also assessed using ImageJ.
Rat anti mouse lamp1
Manufactured by Santa Cruz Biotechnology
The Rat anti-mouse LAMP1 is a primary antibody that specifically recognizes the Lysosome-Associated Membrane Protein 1 (LAMP1) in mouse samples. LAMP1 is a membrane glycoprotein that serves as a marker for lysosomes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse igg1, by BioLegend (1 mentions)
Goat anti rat alexa 568, by Thermo Fisher Scientific (1 mentions)
Goat anti rat alexa488, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat alexa568, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Vector Laboratories (1 mentions)
Alexa 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Lsm 780 microscope, by Zeiss (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phospho ulk1, by Cell Signaling Technology (1 mentions)
Rhodamine red conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti mouse phospho stat1, by Cell Signaling Technology (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Phospho p65, by Cell Signaling Technology (1 mentions)
3 protocols using rat anti mouse lamp1
1
Visualizing Immune Complex Trafficking in Podocytes
2
Podocyte Lysosomal and Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
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WT or FcRn KO podocytes were treated with regular media, anti NP-ova IgG1 antibody (clone N1G9, 20 μg/ml), NP-ova (20 μg/ml) or ICs for 4 or 48 hours. Cell lysates were harvested in RIPA buffer (Thermo Fisher, cat. # 89900) supplemented with phosphatases and proteases inhibitors, subject to western blot analysis and probed with rat anti mouse LAMP1 (Santa Cruz, cat. #sc-19992) or rabbit anti mouse cathepsin B (Cell Signaling, cat. #31718) antibodies. ß-actin (Sigma, cat. # A1978) was used as a loading control. Image J was used to quantitate band intensity.
For signaling studies, WT or FcRn KO podocytes were not treated or treated with NP-ovalbumin, IgG or immune complexes as above for the indicated times. Cell lysates were harvested in RIPA buffer, subject to western blot analysis and probed with rabbit ant mouse AKT (cell Signaling, cat # 4691), rabbit anti mouse phospho-AKT (S473, Cell Signaling, cat. # 4060), rabbit anti mouse ERK1/2 (Cell Signaling, cat. # 4695) or rabbit anti mouse phospho-ERK1/2 (Cell Signaling, cat. # 4377). ß- actin was used as a loading control and band intensity was quantitated using ImageJ.
For signaling studies, WT or FcRn KO podocytes were not treated or treated with NP-ovalbumin, IgG or immune complexes as above for the indicated times. Cell lysates were harvested in RIPA buffer, subject to western blot analysis and probed with rabbit ant mouse AKT (cell Signaling, cat # 4691), rabbit anti mouse phospho-AKT (S473, Cell Signaling, cat. # 4060), rabbit anti mouse ERK1/2 (Cell Signaling, cat. # 4695) or rabbit anti mouse phospho-ERK1/2 (Cell Signaling, cat. # 4377). ß- actin was used as a loading control and band intensity was quantitated using ImageJ.
3
Immunohistochemical Analysis of Mouse Paw
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