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icc #3

Open evaleiyuhang opened 9 months ago

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) ）

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

Immunocytochemistry_SOP (3) (1).pdf

evaleiyuhang commented 9 months ago

16/11/2023 19:37

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) ）

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

"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 day0 Sample preparation. (immunocytochemistry)

  1. Fix cells in \4% paraformaldehyde(\PFA; 25min, RT)（Wear a lab coat, \PFA\ (named FA), clean the table immediately after use, Remove all plate media） ～～24✖️500ul✖️4%PFA（in PBS）=v✖️37%PFA，37%PFA=1297ul，PBS=10721ul ～～～24✖️500ul✖️4%PFA（in PBS）=1297ul 37%PFA + 10721ul PBS
  2. Wash to remove \PFA(3 x 10min washes) with PBS（500ul，3✖️15min，RT）
  3. Add \PBS\ to wells to prevent cells from drying out（500ul）
  4. Seal plate with parafilm and store in fridge until staining.（Edge sealing）

icc day1 21/11/23 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. ～～2/24well✖️（1.2/5）✖️300ul 10% NGS（in 0.3% Triton X-100）=v ✖️100% NGS，100% NGS=90ul，0.3% Triton X-100=900ul ～～900ul 0.3% Triton X-100（in PBS）=v✖️10% Triton X-100，10% Triton X-100=27ul，PBS=900ul- 27ul -90= 783ul ～～～900ul 10% normal goat serum (NGS) in Triton X-100 (0.3% in PBS; PTx; Sigma-Aldrich) = 90ul 100% NGS + 27ul 10% Triton X-100 + 783ul PBS
  2. Stain with primary antibodies against the neuronal/astrocytic antigens of interest in NGS (2%) and PTx (0.06%) at 4⁰C overnight（remove block，300ul，After edge sealing, keep in darkness at 4⁰C overnight. ） [A negative control without primary antibody is used as a control to reveal any nonspecific staining.] ～～2/24well✖️300ul GFAP(chk x-glial fibrillary acidic protein) + β-tubulin（anti-tublin β 3 mouse）（in NGS (2%) and PTx (0.06%)，in PBS）= v✖️block buffer（10% NGS（in 0.3% Triton X-100）），10% NGS（in 0.3% Triton X-100）=120ul，PBS=480ul。 ～～～2/24well✖️300ul primary antibodies GFAP(chk x-glial fibrillary acidic protein) + β-tubulin（anti-tublin β 3 mouse）（in NGS (2%) and PTx (0.06%)，in PBS）=120ul block buffer（10% NGS（in 0.3% Triton X-100））+ 480ul PBS + 1ul GFAP(chicken) + 1ul β-tubulin（anti-tublin β 3 mouse）

ICC Day2 22/11/23 11:00-15:00 🫐🫐🫐🫐

  1. Wash with wash buffer (0.02% PTx; 3 x 10 min washes). （primary antibody recovery. 500ul 0.02% PTx，3 x 10 min） ～～15ml 0.02% PTx（in PBS） = v✖️ 10% PTx，10% PTx = 30ul，PBS=15ml ～～～15ml 0.02% PTx = 15ml PBS + 30ul 10% PTx
  2. Incubate with species-specific secondary antibodies in PTx (0.02%) for 2 h in the dark. （500ul secondary antibodies, dark 2h）(488, goat,不同，mouse一一对应) ～～～2/24well✖️500ul secondary antibodies (in PTx (0.02%) )= 1000ul 0.02% PTx + 1ul 555 goat anti-chicken (chk x-glial fibrillary acidic protein) + 1ul 488 donkey anti-mouse（anti-tublin β 3 mouse）
  3. Wash with wash buffer (3 x 10 min washes).
  4. Incubate cells in Hoechst (1:1000 in PBS, 30 min, RT; Cell Signalling Technology, USA) to counterstain the nucleus. （500ul Hoechst，30 min, RT） ～～2/24well✖️500ul Hoechst (0.1% in PBS）= v✖️ Hoechst， Hoechst=1ul， PBS=1000ul ～～～1000ul Hoechst (1:1000 in PBS）=1ul Hoechst + 1000ul PBS
  5. Wash once with wash buffer to remove Hoechst. （ 500ul wash buffer）
  6. Add PBS (500 μL) to keep the cells hydrated until mounting.（500ul PBS）
  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.（Tags: NAS2, crxastorytes, 22/11/23, GFAP(555 chicken) + β-tubulin(488 mouse), tags 1, 2. Add 5ul of mounting media to the glass slide, use a clamp to take out the cell sheet and turn it upside down so that the cells are in contact with the mounting media. Store it in the dark at room temperature to dry. Original 24-well plate edge-sealed and stored at 4°C.）

imagecupture 23/11/23 14:30-15:30

channel：488，555 exposure：auto ， exposure cvi，tiff save in usb

imagecupture 27/11/23 17:30-18:30

channel：488，555，dapi time：488 time 200us, 555 time 100 us, dapi 75us， exposure cvi，large tiff（full + single channel） save in onedrive