TABLE 3.
Conditions and antibody dilutions optimized using the immunohistochemistry method outlined in this article
| Reference | Target antibody | Clonality | Optimal dilution | Antigen retrieval | Secondary antibody | Optimal dilution |
|---|---|---|---|---|---|---|
| AF markers | ||||||
| [11] a | Collagen typae I (ab90395) | Mouse monoclonal | 1:200 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| [12] b | Collagen type I (ab34710) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| Bone markers | ||||||
| [12] b | Alkaline Phosphatase (ab108337) | Rabbit monoclonal | 1:200 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [12] b | Collagen X (ab49945) | Rabbit polyclonal | 1:400 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [12] b | Osteocalcin (ab13420) | Mouse monoclonal | 1:400 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| [12] b | Osteopontin (ab69498) | Mouse monoclonal | 1:200 | None | Rabbit anti mouse (ab6727) | 1:500 |
| [12] b | Runx2 (ab76956) | Mouse Monoclonal | 1:200 | None | Rabbit anti mouse (ab6727) | 1:500 |
| Cell cycle and apoptosis | ||||||
| [11, 13] a | Caspase 3 (ab13847) | Rabbit polyclonal | 1:400 | None | Goat anti rabbit (ab6720) | 1:500 |
| Unpublished | P16INK4a (ab108349) | Rabbit monoclonal | 1:100 | None | Goat anti rabbit (ab6720) | 1:400 |
| Cell signaling | ||||||
| [14] | c‐jun (ab32385) | Rabbit polyclonal | 1:400 | Heat | Goat anti rabbit (ab6720) | 1:300 |
| [15] c | IHH (Santa Cruz, sc‐1196) | Goat polyclonal | 4ug/mL | Enzyme | Donkey anti goat (ab6884) | 1:400 |
| [9] | NFkB (ab31481) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab6720) | 1:300 |
| [11] | NOTCH2 (ab8926) | Rabbit polyclonal | 1:200 | Heat | Goat anti rabbit (ab6720) | 1:400 |
| [9] | P38 MAPK (ab4822) | Rabbit polyclonal | 1:800 | Heat | Goat anti rabbit (ab6720) | 1:300 |
| [15] c | PTCH (Santa Cruz, sc‐6149) | Goat polyclonal | 1:50 | Heat | Donkey anti goat (ab6884) | 1:300 |
| [15] c | PTHrP (Santa Cruz, sc‐9680) | Goat polyclonal | 1:25 | Heat | Donkey anti goat (ab6884) | 1:300 |
| [15] c | PTHR1 (Santa Cruz, sc‐12 777) | Goat polyclonal | 1:20 | Heat | Donkey anti goat (ab6884) | 1:300 |
| [15] c | SMO (Santa Cruz, sc‐6366) | Goat polyclonal | 1:25 | Heat | Donkey anti goat (ab6884) | 1:300 |
| Cytokines and chemokines | ||||||
| [16] | CCL2 (ab9669) | Rabbit polyclonal | 1:500 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [17] | CCL3 (ab32609) | Rabbit polyclonal | 1:4000 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [17] | CCL4 (ab9675) | Rabbit polyclonal | 1:2000 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [16] | CCL7 (ab104866) | Rabbit polyclonal | 1:10000 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [18] | CCR1(ab89055) | Mouse monoclonal | 1:1000 | Heat | Rabbit anti mouse (ab6727) | 1:400 |
| [16] | CXCL8 (ab7747) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [18] | CXCR1 (ab60254) | Mouse monoclonal | 1:2000 | Heat | Rabbit anti mouse (ab6727) | 1:400 |
| [18] | CXCR2 (ab24963) | Mouse monoclonal | 1:500 | Heat | Rabbit anti mouse (ab6727) | 1:400 |
| [11] a | IL‐1β (ab9722) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [11] a | IL‐1RI (ab100278) | Rabbit polyclonal | 1:100 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [16] | IL‐16 (ab9563) | Rabbit polyclonal | 1:750 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| Immune markers | ||||||
| [18] | CD4 (ab51312) | Mouse monoclonal | 1:500 | Heat | Rabbit anti mouse (ab6727) | 1:400 |
| Unpublished | CD11b (ab62817) | Goat polyclonal | 1:800 | None | Donkey anti goat (ab6884) | 1:500 |
| [12] b | CD68 (ab955) | Mouse monoclonal | 1:200 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| Matrix remodeling | ||||||
| [11] a | ADAMTS4 (ab185722) | Rabbit polyclonal | 1:200 | None | Goat anti rabbit (ab6720) | 1:500 |
| [11] a | MMP3 (ab53015) | Rabbit polyclonal | 1:400 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [11] a | MMP13 (ab39012) | Rabbit polyclonal | 1:200 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| Neural and vascular | ||||||
| [19] | NF200 (ab82259) | Mouse monoclonal | 1:400 | Heat | Rabbit anti mouse (ab6727) | 1:500 |
| [19] | PGP9.5 (ab8189) | Mouse monoclonal | 1:200 | Heat | Rabbit anti mouse (ab6727) | 1:500 |
| [19] | CD31 (ab28364) | Rabbit polyclonal | 1:400 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [20] | Sema3C (ab135842) | Rabbit polyclonal | 1:200 | None | Goat anti rabbit (ab6720) | 1:500 |
| [20] | Sema3D (antibodies online—ABIN1386639) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [20] | NRP‐1 (ab81321) | Rabbit monoclonal | 1:200 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [20] | NRP‐2 (ab185710) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [20] | Plexin A1 (ab32960) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [21] | Substance P (ab10353) | Mouse monoclonal | 1:500 | None | Donkey anti mouse (Alexa Fluor 488) | 1:200 |
| Unpublished | NGF (ab52918) | Rabbit monoclonal | 1:100 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| Unpublished | VEGF (ab52917) | Rabbit monoclonal | 1:100 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| NP markers | ||||||
| [11, 13] a | Aggrecan (ab3778) | Mouse monoclonal | 1:100 | Heat | Rabbit anti mouse (ab6727) | 1:500 |
| [13] a | Chondroitin sulphate (ab11570) | Mouse monoclonal | 1:400 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| [11] a | Collagen type II (ab34712) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [13] a | Collagen type II (ab3092) | Mouse monoclonal | 1:200 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| [10, 21] a | FOXF1 (ab23194) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [11] a | HIF1α (ab16066) | Mouse monoclonal | 1:100 | None | Rabbit anti mouse (ab6727) | 1:500 |
| [22] | KRT‐19 (ab7754) | Mouse monoclonal | 1:400 | None | Rabbit anti mouse (ab6727) | 1:500 |
| [22] | LAM‐5 (ab78286) | Mouse monoclonal | 1:800 | Enzyme | Rabbit anti mouse (ab6727) | 1:500 |
| [10, 21] a | PAX1 (ab203065) | Rabbit polyclonal | 1:400 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| Osmotic regulation | ||||||
| [23] c | AQP0 (ab134695) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [23, 24] c | AQP1 (ab15080) | Rabbit polyclonal | 1:400 | None | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP2 (ab85876) | Rabbit polyclonal | 1:400 | None | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP3 (ab125219) | Rabbit polyclonal | 1:1600 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP4 (ab9512) | Mouse monoclonal | 1:200 | Enzyme | Rabbit anti mouse (ab7074) | 1:500 |
| [23] c [24] | AQP5 (ab92320) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP6 (ab191061) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP7 (ab85907) | Rabbit polyclonal | 1:100 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [23] c | AQP9 (ab85910) | Rabbit polyclonal | 1:400 | Enzyme | Goat anti rabbit (ab6720) | 1:500 |
| [25] | TonEBP (ab3446) | Rabbit polyclonal | 1:100 | Heat | Goat anti rabbit (ab97049) | 1:500 |
| Ca 2+ channels | ||||||
| Unpublished | TRPV1 (ab3487) | Rabbit polyclonal | 1:100 | Enzyme | Goat anti rabbit (ab97049) | 1:500 |
| Unpublished | TRPV4 (ab94868) | Rabbit polyclonal | 1:200 | Enzyme | Goat anti rabbit (ab97049) | 1:500 |
| Lactate transport | ||||||
| [26] | MCT1 (Santa Cruz, sc‐50 324) | Rabbit polyclonal | 1:20 | Heat | Goat anti rabbit (ab97049) | 1:500 |
| [26] | MCT4 (Santa Cruz, sc‐50 329) | Rabbit polyclonal | 1:10 | Heat | Goat anti rabbit (ab97049) | 1:500 |
| [26] | CD147 (Santa Cruz, sc‐9754) | Goat polyclonal | 1:10 | Heat | Donkey anti goat (ab6884) | 1:500 |
Note: All antibodies optimized for use on formalin fixed paraffin embedded (FFPE) human intervertebral disc (IVD) tissue.
a
Publications referenced report these antibodies in optimized for use on human mesenchymal stem cells embedded within hydrogel.
b
Optimized for use on murine bone tissue.
c
Also optimized for use on canine IVD tissue. All antibodies supplied by Abcam unless stated otherwise.