Syndecan 1 (E7F7T) Rabbit mAb #96489

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. Xylene.
2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
3. Deionized water (dH₂O).
4. Hematoxylin (optional).
5. Wash Buffer:
   1. 1X Tris Buffered Saline with Tween^® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween^® 20 (#9997) to 900 ml dH₂0, mix.
6. SignalStain^® Antibody Diluent (#8112).
7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
   1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
   2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
10. Detection System: SignalStain^® Boost IHC Detection Reagents (HRP, Rabbit #8114).
11. Substrate: SignalStain^® DAB Substrate Kit (#8059).
12. Hematoxylin: Hematoxylin (#14166).
13. Mounting Medium: SignalStain^® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:
   1. Incubate sections in three washes of xylene for 5 min each.
   2. Incubate sections in two washes of 100% ethanol for 10 min each.
   3. Incubate sections in two washes of 95% ethanol for 10 min each.
2. Wash sections two times in dH₂O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

1. Wash sections in dH₂O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH₂O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain^® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
7. Equilibrate SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
9. Cover section with 1–3 drops SignalStain^® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
10. Wash sections three times with wash buffer for 5 min each.
11. Add 1 drop (30 µl) SignalStain^® DAB Chromogen Concentrate to 1 ml SignalStain^® DAB Diluent and mix well before use.
12. Apply 100–400 µl SignalStain^® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
13. Immerse slides in dH₂O.
14. If desired, counterstain sections with hematoxylin (#14166).
15. Wash sections in dH₂O two times for 5 min each.
16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
17. Mount sections with coverslips and mounting medium (#14177).

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. 4% Formaldehyde, Methanol-Free (#47746)
3. 100% Methanol (#13604): Chill before use
4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Recommended Anti-Rabbit secondary antibodies::
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 647 Conjugate) #4414
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Fix for 15 min at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
   1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
2. Permeabilize for a minimum of 10 min on ice.
3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.)
2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 min at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

非偶联兔抗体的流式细胞术（活细胞）实验步骤

A. 溶液与试剂

注：使用反渗透去离子水 (RODI) 或同等级别的水配制溶液。

1. 1X 磷酸盐缓冲生理盐水 (PBS)：若要制备 1 L 1X PBS：将 100 ml 10X PBS (#12528) 添加到 900 ml 水，混合。
2. 抗体稀释缓冲液：购买即用型流式细胞术抗体稀释缓冲液 (#13616)，或在 100 ml 1x PBS 中溶解 0.5 g 牛血清白蛋白 (BSA) (#9998) 来配制 0.5 % BSA PBS 缓冲液。4°C 保存。
3. 建议使用抗兔二抗：
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #79408

注：在您的实验中加入荧光细胞染料（包括活力指示染料、DNA 染料等）时，请参考染料产品网页，了解建议的实验步骤。访问 www.cellsignal.com，了解经验证用于流式细胞术的细胞染料完整列表。

B. 免疫染色

注：使用血细胞计数器或备选方法计数细胞。

注：如果使用全血，则需裂解红血细胞，并在免疫染色之前通过离心分离洗涤。

注：人 Fc 受体与兔 IgG 发生交叉反应。当感兴趣的细胞表达高水平的 Fc 受体蛋白（例如，巨噬细胞/单核细胞谱系）时，在用兔抗体进行免疫染色之前，用人 Fc 块预孵育活细胞。

注：最佳离心条件会根据细胞类型和试剂容量变动。一般，1-5 分钟 150-300g 将足以使细胞沉淀下来。

1. 将所需数目的细胞分装入试管或小孔中。（通常，每次测定 5 x 10⁵ 至 1 x 10⁶ 个细胞。）
2. 通过离心使细胞沉淀下来，移除上清液。
3. 在 100 µl 稀释的一抗中重悬细胞，这种一抗按建议的稀释度或如通过滴定所确定那样以抗体稀释缓冲液配制。
4. 在冰上孵育 30 分钟至 1 小时。避光。
5. 在抗体稀释缓冲液或 1X PBS 中通过离心洗涤。弃去上清液。重复。
6. 在 100 µl 稀释的荧光物质偶联的二抗（按建议稀释度用抗体稀释缓冲液制备）中重悬细胞。
7. 在冰上孵育 30 分钟。避光。
8. 用抗体稀释缓冲液进行离心洗涤。弃去上清液。重复。
9. 在 200-500 μl 抗体稀释缓冲液中重悬细胞后用流式细胞分析仪进行分析。