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nas2 astrocycles 下 #5

Open evaleiyuhang opened 9 months ago

evaleiyuhang commented 9 months ago

2个6w，1个freeze。1个split to 4个6w。 【Split 1:4】：remove，+pbs-，+acculase 3min-，+serio 1ml 3次collet 1000rpm 3min，+seriol 1ml resuspend，+250ul/well seriol to coat plate，+750ul serio-EL/well，20ul r.c/well。 【1个6w freeze】：remove，+PBS-，+auuclase 3min-，+seriol-EL 1ml 3次，1000rpm 3min，+cryostro 500ul，收集-80。 【命名】cell line+area+well size+media+date+age：nas2 ctx 1x6w serio 19.9.23 D85

evaleiyuhang commented 9 months ago

acculase，serio-el，cryostro，

evaleiyuhang commented 9 months ago

split 2个6well 到6个6well： 【split】：coat：remove geltex，+seriol el 1.7ml，+300ul cells，+r.c20ul。Collect：remove，+PBS-，+auuctate 3min，+seriol 1000rpm 3min，resupend 1ml，300ul/well，3个6w

evaleiyuhang commented 9 months ago

feed：6个6w。【feed】Remove，+SERIOL EL 6ml。【tips】（每天2ml）

evaleiyuhang commented 9 months ago

feed，6个6w：【feed】：remove，+SERIOL EL 3ml/per well

evaleiyuhang commented 9 months ago

【tips】mature D90 changeEGF+HLF to BMP4+HLF

evaleiyuhang commented 9 months ago

"mature：24个24w，【mature】+seriol no EL 600ul，+BMP4 6ul，+LIF 6ul（600ul个24w=1440ul=15ml（15ml SERIOL，15ul BMP4，15ul LIF））：remove，+600ul perwell
【tips】 D90 changeEGF+HLF to BMP4+HLF"

evaleiyuhang commented 9 months ago

mature feed：24个24w，【mature feed】-all remove，+500ul seriol no EL（24孔板24孔）

evaleiyuhang commented 9 months ago

watch

evaleiyuhang commented 9 months ago

"PFA: 24✖️24w，clean with \4% PFA\ 500ul，friege 【calculate】： \4%PFA\：24/24w✖️500ul=12000ul， 、\37%PFA\ to 12000ul \4%PFA\：12000ul/37/4=1297ul， 、\PBS\：12000-1297ul \37% PFA\=10721ul 、10721ul \PBS+1297ul \37%PFA\=12000UL \4%PFA\ 【Protocol】

  1. 10721ul \PBS+1297ul \37%PFA\=12000UL\ 4%PFA\（穿实验服，\PFA\（名叫FA），用完立即清洗桌子）
  2. Remove all plate media
  3. +\4%PFA\ 500ul/w（吹打混匀），15min，RT，remove\PFA\（标记PFA FT为wast）
  4. +\PBS\ 500ul，3x10min（可省10min，直接清洗）（直接一次添加清洗）
  5. +\PBS\ 500ul
  6. 封闭，存储在冰箱

Sample preparation. (immunocytochemistry)

  1. Fix cells in \4% paraformaldehyde(\PFA\; 25min, RT)
  2. Wash to remove \PFA(3 x 10min washes) with PBS
  3. Add \PBS\ to wells to prevent cells from drying out Seal plate with parafilm and store in fridge until staining. 下周继续"
evaleiyuhang commented 9 months ago

"icc day1 11:30-15:00 🫐

Immunostaining protocol

  1. Block cells with 10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich) for 2 h. （300ul/well，之前都是加500ul，10% PTx在adina桌子上，PBS是tablet+超纯水配置的也在桌子上） 5孔 x 300ul= 1500ul（制备3000ul 10% normal goat serum (NGS) in Triton X-100，需要制备下一步多一点，100ul即可？？） 3ml 10% normal goat serum (NGS) =3ml / 100/10%=300ul normal goat serum (NGS) 3ml Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich)= 3ml / 10% / 0.3% =90ul 10% Triton X-100 3ml Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich)=3ml-300 ul normal goat serum (NGS)-90ul 10% Triton X-100=2610ul PBS 3ml 10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich)=300ul normal goat serum (NGS) + 90ul 10% Triton X-100 + 2610ul PBS
  2. Stain with primary antibodies against the neuronal/astrocytic antigens of interest in NGS (2%) and PTx (0.06%) at 4度 overnight（抗体在进门靠桌子的冰箱下层，一个一个找，记得最后primary antibody扔掉的时候要回收。加抗体之前记得混匀一个一个加，封边后，黑暗状态，放桌面上常温过夜） [A negative control without primary antibody is used as a control to reveal any non- specific staining.] Astrocyte marker：GFAP(chicken), SlOOb(mouse). Cortical marker：Tbr1(rabbit). Midbrain marker：LMX1A(rabbit), FOXA2(mouse), SOX6(rabbit),aldh1a1(mouse) 1 GFAP(chicken) FOXA2(mouse) 2 GFAP(chicken) aldh1a1(mouse) 3 SlOOb(mouse) LMX1A(rabbit) 4 SlOOb(mouse) Tbr1(rabbit) 5 SlOOb(mouse) SOX6(rabbit) NGS (2%) and PTx (0.06%) 是 block buffer 的五倍，所以直接在上一步多加一些。 500ul NGS (2%) and PTx (0.06%) = 500ul /10%/2%=100ul block buffer（10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich) ） 500ul primary antibodies= 500ul -100ul block buffer（10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich) = 400ul PBS 500ul primary antibodies= 0.5ul + 0.5ul + 400ul PBS + 100ul block buffer（10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich) ） "
evaleiyuhang commented 9 months ago

"ICC Day2 08:00-14:00 🫐🫐🫐🫐

  1. Wash with wash buffer (0.02% PTx; 3 x 10 min washes). （primary antibody 收集，存放在进门靠桌子冰箱最下层，加500ul 0.02% PTx，3次每次10min洗涤） 50ml 0.02% PTx=50ml/ 10%/0.02%=0.1ml 10% PTx 50ml 0.02% PTx = 50ml PBS + 100ul 10% PTx（10% PTx在adina桌子上，PBS是tablet+超纯水配置的也在桌子上）
  2. Incubate with species-specific secondary antibodies in PTx (0.02%) for 2 h in the dark. （chicken在进门靠桌子的冰箱，其余的在进门的冰箱下层盒子里，添加500ul secondary antibodies，黑暗下2h） 1 GFAP(chicken) FOXA2(mouse) Chicken+mouse 2 GFAP(chicken) aldh1a1(mouse) Chicken+mouse 3 SlOOb(mouse) LMX1A(rabbit) Mouse+rabbit 4 SlOOb(mouse) Tbr1(rabbit) Mouse+rabbit 5 SlOOb(mouse) SOX6(rabbit) Mouse+rabbit 2个 x 500ul= 1ml chicken-mouse secondary antibodies 1ml chicken-mouse secondary antibodies = 0.5ul chicken secondary antibodies+ 0.5ul mouse secondary antibodies + 1ml PTx (0.02%) 3个 x 500ul= 1.5ml mouse-rabbit secondary antibodies 1.5ml mouse-rabbit secondary antibodies = 1.5ul mouse secondary antibodies+ 1.5ul rabbit secondary antibodies + 1.5ml PTx (0.02%)
  3. Wash with wash buffer (3 x 10 min washes). （PTx (0.02%)一个一个洗，3次10min）
  4. Incubate cells in Hoechst (1:1000 in PBS, 30 min, RT; Cell Signalling Technology, USA) to counterstain the nucleus. （Hoechst位于进门靠桌子冰箱的下层在抗体那格） 5个 x 500ul= 2.5ml Hoechst (1:1000 in PBS） 2.5ml Hoechst (1:1000 in PBS） = 2.5ml / 1000/1=2.5ul Hoechst 2.5ml Hoechst (1:1000 in PBS） = 2.5ml PBS 2.5ml Hoechst (1:1000 in PBS）=2.5ul Hoechst + 2.5ml PBS
  5. Wash once with wash buffer to remove Hoechst. （用PTx (0.02%)洗一次，不用10min？）
  6. Add PBS (500 μL) to keep the cells hydrated until mounting.
  7. Mount coverslips on slides in mounting media (5 μL; 1:1 PBS-glycerol), seal with clear nail varnish and stored at 40C in the dark. （mounting media位于进门冰箱下层门上，拨片在maddie桌子上二层，夹子在桌子旁冰箱的抽屉里，夹子取出细胞片倒扣让细胞与mounting media接触，存放在室温黑暗状态使其干燥。原始24孔板封边4度存放。） 先标记，na2s，crxastorytes，18.10.23，抗体+抗体，标记1，2， 加5ul mounting media在玻片上，用夹子取出细胞片倒扣让细胞与mounting media接触，存放在室温黑暗状态使其干燥， 原始24孔板封边，4度存放。

"

evaleiyuhang commented 9 months ago

"4度存放拨片 18:00-18:01🥥

黑暗状态下干燥后的拨片存储在盒子里，放置在进门冰箱下层"

evaleiyuhang commented 9 months ago

"显微镜培训14:00-15:00🍎

Bioimaging - Laser Scanning Confocal Microscope (LSM800) 生物成像 - 激光扫描共焦显微镜 (LSM800)

团队教学 两个按钮，1.5小时休息， 日历记录， 数量内容

软件： zenblo---左按钮--calablite now--

观察： 活体自我曝光（一般是246.229ms，如果太高会不真） d盘放置活体 ，继续这台没用区别 就关掉激光灯，长时间对样本会损伤。

拍摄： 采集方式--智能设置--设置三个设备 显微镜光学原理？ z-stack- 设备+z-stack 尺寸： 采集方式--位深：12？ 显示（右下角，调节，图像好，是一样的） ） 信息是指，详细信息 图：加标尺

保存为： Cz D-user-lab 上传googledrive "

evaleiyuhang commented 9 months ago

"Split astrocycles 20ml 小瓶子 1:4 下层 16/11/2023 18:06 🥥 ·～1 small bottle 1:4 4 small bottle 20ml小瓶子，coat with geltex 2ml wait at least 2h Remove all media in one bottle，wash with PBS 1滴管，+EDTA 1滴管 4min - Remove geltrax in 4 coat bottle，add 2滴管 SERIO 命名（NAS2 umAPC NA D92 D0 21/4/23 16/11/23 D92） Collect cells with SERIO 2滴管，split to 4 coat bottle 拧紧瓶口后松开一点点 酒精消毒下层恒温箱"