Evans Blue Stain (2%) 伊文思蓝染色液（2%）

产品信息

产品描述

伊文思蓝（Evans Blue，CAS NO：314-13-6），也称作埃文斯蓝，是一种非膜渗透性的染料。当质膜受损，染料能进入细胞质和细胞核，从而将其染成蓝色，可用来检测细胞活力。伊文思蓝还能用来研究血脑屏障（BBB）渗透性，通过与白蛋白结合来指示血脑屏障穿透蛋白能力。正常情况下，血浆白蛋白不能透过血脑屏障，所以染色后具完整血脑屏障的神经系统不着色，而完整性被破坏的神经系统会着色。

本品为2%的伊文思蓝染色液，根据具体用途直接使用或简单稀释后再使用。

保存与运输方法

保存：2-8℃避光保存，1年有效。

运输：室温运输。

使用方法

根据具体实验用途，直接用伊文思蓝染色液（2%）或将其用PBS稀释到伊文思蓝染色液（0.5%）再使用。

以下染色步骤以伊文思蓝染色液（0.5%）为例，仅做参考。

一、血脑屏障通透性

一.1 取处理后的动物（以小鼠为例），经尾静脉或股静脉按照2~3ml/kg体重的比例注射伊文思蓝染色液（0.5%） 数s至1min内，小鼠眼睛、皮肤出现蓝色。0.5～1h 后处死小鼠，取出目的脑组织。【注意】：伊文思蓝染色液的注射用量需要根据动物类型以及体重来决定。

一.2 将脑组织置于 1.5ml 离心管中，加入 1ml PBS，迅速用组织匀浆器将脑组织制成匀浆，4℃ 1000g离心15min。

一.3 取上清，加入等量三氯*-乙酸，4℃孵育18~24h。该步骤亦可采用如下操作：取上清，按上清：丙酮=3:7比例加入丙酮，室温孵育24h。

一.4 4℃ 1000g离心20~30min（或2000g离心15min）。

一.5 取上述溶液1~2ml，用分光光度计测 620 nm下的OD值。同时测定已知不同梯度的标准伊文思蓝的OD值，绘制标准曲线。根据标准曲线计算出待测样品的伊文思蓝含量。

二、细胞活力鉴定

2.1 取 100μl 重悬细胞到常规离心管内，加入100μl 伊文思蓝染色液（0.5%）轻轻混匀，染色3min(染色时间可适当延长，但不宜超过 10min)。

2.2吸取少量经过染色后的细胞，用血细胞计数板计数。通常如果要比较精确地进行定量，每个细胞样品至少数500个细胞，数出蓝色细胞和细胞总数。

三、种子染色

3.1 用刀片做横切和沿种胚中央准确纵切，入染色液染色3~5min。

3.2 蒸馏水中浸泡20~60min，视脱色程度而定。

注意事项

1） 伊文思蓝对人体有轻微毒性，操作过程中请注意防护。

2） 细胞染色时，需注意凋亡小体偶尔也有拒染现象。

3） 建议用低温冷冻离心机进行离心。

4） 为了您的安全和健康，请穿实验服并戴一次性手套操作。

相关产品

文献示例（An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse，Ref：PMID: 30272649）

This method uses FVBN adult mice, aged 16 - 20 weeks, found to be optimal for the purposes of this study. Day 1 includes steps 1 - 5 and Day 2 includes steps 6 - 7 (Figure 1).

1. Equipment Preparation

1. Secure an adequate supply of sterile, disposable syringes and needles, if ketamine/xylazine is used as the anesthetic (as recommended). If isoflurane is used as the anesthetic, check the oxygen tank and the fluid level of isoflurane to make sure there are adequate supplies for the experiment before starting. Also, assemble the nosecone breathing circuits and attach them to the induction box; attach new charcoal canisters to the breathing circuits. Prepare the induction box by turning on the oxygen and ascertaining that the second stage reads approximately 50 psi.

2. Turn on the heating pad to 37 °C.

3. Prepare the rectal temperature probe for the surgery.

2. Mouse Preparation

This step includes anesthesia, hair removal, and positioning (adult FVBN mice-age 16 - 20 weeks).

1. Weigh the mice and record the weights.

2. Anesthetize the mice.

1. Administer ketamine and xylazine IP (80 - 100 mg/kg and 7.5 - 16 mg/kg, respectively). However, it is recommended to begin with lower doses of ketamine and xylazine (about 30 mg/kg and 6 mg/kg, respectively).

2. Maintain the anesthesia with about 0.1 - 0.25 times initial doses of ketamine/xylazine throughout the surgery. Ketamine and xylazine were the anesthetic agent(s) of choice in this particular study, as more reproducibility and survivability were observed. The anesthetic agent(s) of choice in other studies may be found to be different.  If isoflurane is used, put the mouse in an induction chamber and turn on isoflurane to 5% until the mouse loses full consciousness. Then use a nasal nosecone set at 1.5 - 2.5% isoflurane throughout the remainder of the procedure.

3. Monitor the mice every 2 - 3 min by toe pinch to check for appropriate depth of anesthesia.

4. Shave the ventral neck area of the mouse.

5. Place the mouse in a supine position on the preheated pad. Secure the paws and feet of the mouse to the surgical surface with tape.

6. Place artificial tear ointment onto the eyes to prevent drying out during surgery.

3. Surgical Details

1. Make a 1 cm incision in the right ventral neck over the jugular vein.

2. Apply one or two drops of lidocaine (1 - 2%) into the incision area for pain management and to promote vasodilation. Wait 2 min. for the lidocaine to take effect.

3. Expose and isolate the right internal jugular vein via blunt dissection. Tie the vein off with 4-0 suture and gently retract the rostral end of the vessel with a hemostat. Cut a hole, using fine scissors, about 3 mm below or caudal to the tie, approximately halfway through the diameter of the vein.

4. Mark a PV-1 polyvinyl catheter 1.5 cm from the end and insert it into the jugular vein through the hole using a vessel dilator, and thread the catheter towards the caudal end of the vessel, approximately 1.5 cm.

5. Tie the catheter securely within the caudal part of the vessel (below the cut), with 4-0 silk. Tie the rostral part of the vessel to the outside of the catheter with the loose ends of the suture which was used to tie off the rostral jugular vein, in step 3.3.

6. Tack the skin loosely back together around the catheter with 4-0 silk to help prevent loss of body heat and desiccation of tissues.

7. Connect the catheter to a syringe containing heparinized saline (10 U/mL) and flush the catheter.

4. Injections

1. Inject the Evans blue solution (50 μL of a 30 mg/mL solution in 0.9% normal, unheparinized saline, or approximately 50 mg/kg) into the jugular vein catheter, followed by a small volume of heparinized saline to flush the line.

2. 2 min later, inject substance P (100 μL of a 0.3 μM solution in 0.9% normal, unheparinized saline, or 1 nmol/kg), followed by a small volume of heparinized saline to flush the line. Substance P augments the extravasation of plasma proteins through the endothelial layer; in this protocol, it routinely induces an augmentation of plasma extravasation values of approximately 1.5-fold, making plasma extravasation values easier to measure.

3. Wait 18 min after the substance P is injected. During this time, the Evans blue dye will equilibrate and circulate.

4. Terminate the experiment 18 min after the injection of substance P (20 min after Evans blue injection) by sacrificing the mouse with cervical dislocation. It is likely that the mouse can be directly cervically dislocated, without first giving an overdose of anesthetic, as the mouse is probably still well-anesthetized from the surgery. However, if it is necessary, an overdose of the anesthetics ketamine/xylazine, isoflurane, or pentobarbital may be given, followed by cervical dislocation.

5. Isolation of Organs

1. Cut open the chest cavity of the mouse and gravity-perfuse (from a height of about 51 cm or 20") the heart and blood vessels with 50 mL of 50 mM sodium citrate, pH 3.5. pH 3.5 presumably preserves Evans blue binding to albumin.

2. Excise 1 - 5 relevant organs (tissues) with a dissecting scalpel (e.g. urinary bladder, kidney, stomach, liver, pancreas, proximal or distal colon, ileum, duodenum, flank skin, ears, tail, heart, and/or lungs) and remove any residual contents, if present.

3. Rinse the organs in room temperature (RT) phosphate-buffered saline (PBS; 1.44 g of Na₂HPO4, 0.24 g of KH₂PO4, 8.0 g of NaCl, and 0.20 g of KCl in 1 L, pH 7.4).

4. Blot the organs with tissue, cut each organ in half, and weigh each half (wet weights, in g).

5. Dry one half of the tissue in a drying oven at 150 °C, on foil, for 48 h.

6. Place the other tissue half in a consistent volume (up to 200 µL) of formamide in a microfuge tube for 48 h (and up to 72 h) to extract the Evans blue.

6. Measurement of Tissue OD

1. Remove 50 µL of Evans blue-infused formamide (after 48 - 72 h RT incubation) from the microfuge tube and place into one well of a 96-well polystyrene plate. Be careful not to transfer tissue pieces along with the formamide.

2. Place 50 µL of new, pure formamide into each of two empty wells of the 96-well plate for the blanks.

3. Measure and record the OD₆₂₀ of each well of the 96-well plate on an absorbance plate reader. 620 nm is the absorbance max of Evans blue.

7. Calculation of Plasma Extravasation

1. Weigh the dry tissue half which has been in the oven for 48 h.

2. Calculate the wet weight/dry weight ratio for the specific organ of interest from each individual mouse, starting with the wet weight of this tissue half (obtained in step 5.4), divided by the dry weight of this same tissue half (obtained in step 7.1).

3. Calculate the dry weight (in g) of the tissue half in formamide by dividing the wet weight of the tissue half before it was placed in formamide (obtained in step 5.4) by the wet: dry weight ratio for the specific organ of interest (calculated in step 7.2).

4. Calculate the corrected OD₆₂₀ values. Starting with the OD₆₂₀ value from each experimental well of the 96-well plate (containing Evans blue-infused formamide from each tissue, obtained in step 6.3), subtract the blank well OD₆₂₀ value (the mean OD₆₂₀ value of the two wells containing pure formamide, prepared in step 6.2, OD₆₂₀ values obtained in step 6.3) from each experimental value.

5. Calculate the plasma extravasation value by dividing the corrected OD₆₂₀ (calculated in step 7.4) by the dry weight of the tissue half in formamide (calculated in step 7.3). The units of plasma extravasation will be OD₆₂₀/g dry weight.

6. Analyze data and express as the mean ± SEM. Statistically compare groups by t test or one-way analysis of variance and Scheffé's multiple-comparison test (lycofs01.lycoming.edu), as appropriate.

— —Written/Edited by V. Shallan【版权归MKBio懋康所有】

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伊文思蓝染色液 指示剂

伊文思蓝染色液 指示剂