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Anti-HA [12CA5] in Immunocytochemistry (Human)

Addgene #194541

Data Submitted By: Katherine Nardone
Lab Name: Addgene
Submission Date: October 02, 2024
Publication Date: October 08, 2024 (modified October 09, 2024)
Abstract: Experimental results for Anti-HA [12CA5] (Addgene #194541) in immunocytochemistry assay with a human sample.

HeLa cells were untransfected or transiently transfected with pLenti puro HA-H2BC21 (Addgene #227589) and labeled 96 h later with Anti-HA [12CA5]. — Caption: HeLa cells were untransfected or transiently transfected with pLenti puro HA-H2BC21 (Addgene #227589) and labeled 96 h later with Anti-HA [12CA5].
Image attribution: Katherine Nardone, Addgene

This report is made available under the Creative Commons Attribution 4.0 International License.

Antibodies Used: Anti-HA [12CA5]
Addgene #194541; Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594
Thermo Fisher #A-1105

Materials & Methods

Sample

Species: Human
Cell / Tissue Type: HeLa
Additional Information: HeLa cells were untransfected or transiently transfected with pLenti puro HA-H2BC21 (Addgene #227589) and labeled 96 h later with Anti-HA [12CA5].

Buffer

Name: PBS

Fixative

Fixative: Formaldehyde/Paraformaldehyde
Time: 15 min
Temperature: On ice

Permeabilization

Agent: 0.2% Triton X-100 in PBS
Time: 10 min
Temperature: Room Temperature

Blocking

Agent: BSA
Concentration: 5%
Time: 20 min
Temperature: Room Temperature

Primary Antibody

Name: Anti-HA [12CA5]
Source: Addgene
Catalog Number: 194541
Species: Mouse
Gene / Target: Hemaglutinin (HA)
Concentration: 1 µg/mL
Time: 90 min
Temperature: Room Temperature

View in Addgene Catalog

Secondary Antibody

Name: Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594
Source: Thermo Fisher
Catalog Number: A-1105
Concentration: 2 µg/mL
Time: 30 min
Temperature: Room Temperature

Additional Information

Protocol: Addgene immunocytochemistry protocol

Results

Result: Pass (The antibody worked under these conditions)
Describe Results: Antibody #194541 successfully labeled HA-tagged histones in HeLa cells. With a DAPI stain applied, co-localization of the stain along with antibody fluorescence shows the Anti-HA [12CA5] bound to the nuclear region of transfected cells.