Immunohistochemistry Validation

• Antibody Validation
• Flow Cytometry Validation
• Immunofluorescence Validation
• Antibodies Validated for Immunohistochemistry

Activation State-specific and Total Protein Antibodies Validated for Immunohistochemistry

The determination of target specificity in immunohistochemical (IHC) analysis requires multiple validation steps. Cell Signaling Technology’s (CST) in-house IHC group uses a variety of approaches for each and every antibody validated for use in IHC to demonstrate that staining achieved with the antibody is specific and believable. The purpose of this article is to highlight select experiments on various targets and does not aim to show all experiments performed on any particular target.

Western Blot Analysis

All of CST’s antibodies are initially tested by Western blot, and only antibodies that yield clear bands of the appropriate molecular weight with no additional cross-reacting bands are chosen for validation in applications such as immunohistochemistry.

Paraffin-embedded Cell Pellets

Cells are subjected to treatments known to induce signaling changes to verify modification specificity (e.g. phosphorylation, acetylation, cleavage, etc.). To determine phospho-specificity of Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060, LNCaP cells were treated with the PI3K inhibitor LY294002, which dramatically reduced signal as compared to the control, consistent with an expected loss of phosphorylation with the treatment (figure 1). Cell lines known to express or lack expression of the target of interest are used to verify specificity of antibodies recognizing total protein. Alternatively, cells can be treated to detect anticipated changes in localization of the target as compared to control, or siRNA can be used to block expression of the target. In figure 1, treatment of LNCaP cells with LY294002 shifted staining from the membrane to the cytoplasm as detected using Akt (pan) (C67E7) Rabbit mAb #4691.

Phosphatase Treatment

Treatment with phosphatase is used as an additional test to verify phospho-specificity of the antibody. As shown in figure 2, signal obtained using Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060 was completely ablated after treatment with phosphatase.

Blocking Peptides

Peptide blocking verifies sequence specificity of the phospho-specific or total antibody and rules out Fc-mediated binding, biotin background and other non-specific staining.

Tissue Array

Having stained cells consistently and appropriately in models with known target expression levels, antibodies can then be applied to arrays of human cancer tissues to assess performance over a broad spectrum of tissue types.

Xenografts

Xenografts generated from cell lines with known target expression levels are a valuable tool to verify target specificity of phospho-specific and total protein antibodies. Furthermore, when combined with a previously characterized drug treatment, these models can be used to show target modulation upon treatment. For example, gefitinib treatment substantially reduced phospho-p44/42 MAPK (Erk1/2) and phospho-S6 staining as compared to control xenograft using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370 and Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit mAb #4858 (figure 3).

Mouse Models of Cancer

CST routinely verifies antibody performance in relevant mouse models of cancer. As shown in figure 4, tissues from WT and PTEN (-/-) mouse prostate were used to assess staining achieved using Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060. The PTEN/protein/lipid phosphatase is a negative regulator of the PI3K/Akt pathway that is often mutated or absent in various cancers. IHC analysis of the prostate from PTEN (-/-), but not WT mice shows strong staining using Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060 indicating that the staining is specific and due to the lack of PTEN in the PTEN (-/-) mice.

Apc (Min/+) mouse intestinal adenoma was used to assess staining obtained with β-Catenin Antibody (Carboxy-terminal Antigen) #9587 (figure 5). This mouse strain is highly susceptible to spontaneous intestinal adenoma formation due to a mutation in adenomatous polyposis coli (APC). In the normal intestine, β-catenin resides at the membrane at adherens junctions; APC functions as a negative regulator of Wnt signaling by contributing to the destabilization of cytosolic β-catenin. In the intestinal adenoma, the cytoplasmic pool of β-catenin is stabilized and transported to the nucleus. Significant nuclear and cytoplasmic staining was observed in Apc (Min/+) mouse intestinal adenoma using β-Catenin Antibody (Carboxy-terminal Antigen) #9587, consistent with diminished APC.

Staining on Frozen Tissue

CST routinely validates antibodies for staining on fresh frozen tissues. Multiple fixatives are used to determine which fixative is optimal for a given antibody. In figure 6, formaldehyde, formaldehyde/methanol and 10% NBF were compared side by side on frozen U-87MG xenograft using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit mAb #4858 showing that optimal staining was achieved using NBF.

Figure 6 IHC analysis of frozen U-87MG xenograft using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit mAb #4858. Formaldehyde (left), formaldehyde/methanol (middle), and NBF fixation (right) were compared side by side to determine optimal fixation protocol.