Anti-LAMP1 抗体 [EPR4204] - Lysosome Marker

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human liver tissue staining CTBS with ab322065 at a 1 : 2000 (0.249 ug/ml) dilution, ab108597 anti-LAMP1 used at 1 : 400 (0.37 ug/ml) dilution.

Panel A : merged staining of anti-CTBS (green; Opal™520) and anti-LAMP1 (magenta; Opal™570) on human liver.
Panel B : anti-CTBS showed granular staining in human liver.
Panel C : anti-LAMP1 showed granular staining in human liver.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab322065 and ab108597 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human colon tissue staining CTBS with ab322065 at a 1 : 2000 (0.249 ug/ml) dilution, ab108597 anti-LAMP1 used at 1 : 400 (0.37 ug/ml) dilution.

Panel A : merged staining of anti-CTBS (green; Opal™520) and anti-LAMP1 (magenta; Opal™570) on human colon.
Panel B : anti-CTBS showed granular staining in human colon.
Panel C : anti-LAMP1 showed granular staining in human colon.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab322065 and ab108597 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Unknown

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

All lanes:

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (ab108597) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

JAR cell lysate at 10 µg

Lane 3:

Jurkat cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Predicted band size: 45 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver carcinoma tissue sections labeling LAMP1 with purified ab108597 at 1/400 dilution (0.36 μg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody.

Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Immunohistochemical analysis of LAMP1 in paraffin embedded Human kidney tissue, using unpurified ab108597 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

LAMP1 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID : 27061067, 15111122).

All lanes:

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (ab108597) at 1/5000 dilution

Lane 1:

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

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 20µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 20µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate 20µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 110 kDa

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Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Lanes 1 - 4 : Merged signal (red and green). Green - unpurified ab108597 observed at 110 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab108597 was shown to recognize LAMP1 in wild-type HAP1 cells as signal was lost at the expected MW in LAMP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and LAMP1 knockout samples were subjected to SDS-PAGE. ab108597 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (ab108597) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

LAMP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 whole cell lysate at 20 µg

Lane 4:

Human Kidney whole cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (AB108597)

Western blot : Anti-LAMP1 antibody [EPR4204] - Lysosome Marker staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab108597 was shown to bind specifically to LAMP1. A band was observed at 100-120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in LAMP1 knockout cell line. To generate this image, wild-type and LAMP1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LAMP1 antibody [EPR4204] - Lysosome Marker (ab108597) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

LAMP1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Observed band size: 100-120 kDa

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